U.S. patent number 5,800,992 [Application Number 08/670,118] was granted by the patent office on 1998-09-01 for method of detecting nucleic acids.
Invention is credited to William J. Dower, Stephen P.A. Fodor, Dennis W. Solas.
United States Patent |
5,800,992 |
Fodor , et al. |
September 1, 1998 |
Method of detecting nucleic acids
Abstract
The present invention provides method and apparatus for
sequencing, fingerprinting and mapping biological macromolecules,
typically biological polymers. The methods make use of a plurality
of sequence specific recognition reagents which can also be used
for classification of biological samples, and to characterize their
sources.
Inventors: |
Fodor; Stephen P.A. (Palo Alto,
CA), Solas; Dennis W. (San Francisco, CA), Dower; William
J. (Menlo Park, CA) |
Family
ID: |
27496811 |
Appl.
No.: |
08/670,118 |
Filed: |
June 25, 1996 |
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
168904 |
Dec 15, 1993 |
|
|
|
|
624114 |
Dec 6, 1990 |
|
|
|
|
362901 |
Jun 7, 1989 |
|
|
|
|
492462 |
Mar 7, 1990 |
5143854 |
|
|
|
Current U.S.
Class: |
506/9; 435/6.12;
506/16; 536/24.3 |
Current CPC
Class: |
B01J
19/0046 (20130101); B82Y 10/00 (20130101); B82Y
30/00 (20130101); C07C 229/14 (20130101); C07C
229/16 (20130101); C07D 263/44 (20130101); C07D
317/62 (20130101); C07H 19/04 (20130101); C07H
19/10 (20130101); C07H 21/00 (20130101); C07K
1/042 (20130101); C07K 1/045 (20130101); C07K
1/047 (20130101); C07K 7/06 (20130101); C07K
17/06 (20130101); C07K 17/14 (20130101); C12Q
1/6809 (20130101); C12Q 1/6816 (20130101); C12Q
1/6827 (20130101); C12Q 1/6834 (20130101); C12Q
1/6837 (20130101); C12Q 1/6874 (20130101); G01N
21/6428 (20130101); G01N 21/6452 (20130101); G01N
21/6458 (20130101); G03F 7/00 (20130101); G03F
7/265 (20130101); G03F 7/38 (20130101); G11C
13/0014 (20130101); G11C 13/0019 (20130101); C12Q
1/6809 (20130101); C12Q 1/6874 (20130101); C12Q
1/6834 (20130101); B01J 2219/00315 (20130101); B01J
2219/00432 (20130101); B01J 2219/00434 (20130101); B01J
2219/00436 (20130101); B01J 2219/00459 (20130101); B01J
2219/00468 (20130101); B01J 2219/00475 (20130101); B01J
2219/005 (20130101); B01J 2219/00527 (20130101); B01J
2219/00529 (20130101); B01J 2219/00531 (20130101); B01J
2219/00585 (20130101); B01J 2219/0059 (20130101); B01J
2219/00596 (20130101); B01J 2219/00605 (20130101); B01J
2219/00608 (20130101); B01J 2219/0061 (20130101); B01J
2219/00612 (20130101); B01J 2219/00617 (20130101); B01J
2219/00621 (20130101); B01J 2219/00626 (20130101); B01J
2219/0063 (20130101); B01J 2219/00637 (20130101); B01J
2219/00644 (20130101); B01J 2219/00648 (20130101); B01J
2219/00659 (20130101); B01J 2219/00689 (20130101); B01J
2219/00695 (20130101); B01J 2219/00711 (20130101); B01J
2219/00722 (20130101); B01J 2219/00725 (20130101); C07B
2200/11 (20130101); C40B 40/06 (20130101); C40B
40/10 (20130101); C40B 60/14 (20130101); G01N
15/1475 (20130101); C12Q 2535/131 (20130101); C12Q
2535/131 (20130101); C12Q 2565/626 (20130101); C12Q
2563/149 (20130101) |
Current International
Class: |
B01J
19/00 (20060101); C07B 61/00 (20060101); C07H
21/00 (20060101); C07H 19/10 (20060101); C07H
19/00 (20060101); C12Q 1/68 (20060101); G03F
7/26 (20060101); G03F 7/00 (20060101); G11C
13/02 (20060101); G01N 15/14 (20060101); C12Q
001/68 (); C07H 021/02 (); C07H 021/04 () |
Field of
Search: |
;435/6 ;536/24.3
;935/77,78 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
392 546 |
|
Oct 1990 |
|
EP |
|
2233654 |
|
Jan 1991 |
|
GB |
|
WO 89/11548 |
|
Nov 1989 |
|
WO |
|
WO 89/10977 |
|
Nov 1989 |
|
WO |
|
WO 90/00626 |
|
Jan 1990 |
|
WO |
|
WO 90/03382 |
|
Apr 1990 |
|
WO |
|
WO 90/04652 |
|
May 1990 |
|
WO |
|
WO 90/15070 |
|
Dec 1990 |
|
WO |
|
WO 91/07087 |
|
May 1991 |
|
WO |
|
WO 92/10092 |
|
Jun 1992 |
|
WO |
|
WO 92/10588 |
|
Jun 1992 |
|
WO |
|
WO 93/17126 |
|
Sep 1993 |
|
WO |
|
Other References
Perkin Elmer Cetus, GeneAmp DNA Amplification Reagent Kit, insert,
Oct. 1988. .
Church et al, Proc. Natl. Acad. Sci., 81:1991-1995 (Apr., 1984).
.
Ekins et al, Analytica Chimica Acta, 227:73-96 (1989). .
Amit et al., "Photosensitive protecting groups of amino sugars and
their use in glycoside synthesis. 2-nitrobenzyloxycarbonylamino and
6-nitroveratryloxy-carbonylamino derivatives" J. Org. Chem. 39(2)
:192-196 (1974). .
Bains and Smith, "A novel method for nucleic acid sequence
determination," J. Theor. Biol. 135: 303-307 (1988). .
Barinaga, M., "Will `DNA Chip` Speed Genome Initiative?" Science
253:1489 (Sep. 27, 1991). .
Carrano et al., "A high-resolution, fluorescence-based,
semiautomated method for DNA fingerprinting" Genomics 4:129-136
(1989). .
Chetverin, A.B. and Kramer, F.R., "Oligonucleotide Arrays: New
Concepts and Possibilities" Bio/Tech. 12:1093-1099 (Nov. 1994).
.
Coulson et al., "Toward a physical map of the genome of the
nematode Caenorhabditis elegans" Proc. Natl. Acad. Sci. USA
83:7821-7825 (Oct. 1986). .
Craig et al., "Ordering of cosmid clones covering the herpes
simplex virus type I (HSV-I) genome: A test case for fingerprinting
by hybridisation," Nucl. Acids Res. 18:2653-2660 (1990). .
Dower, W.J. and Fodor, S., "The Search for Molecular Diversity
(II): Recombinant and Synthetic Randomized Peptide Libraries" Ann.
Rep. Med. Chem. 26:271-280 (1991). .
Drmanac et al., "Reliable hybridization of oligonucleotides as
short as six nucleotides" DNA Cell Biol. 9(7): 527-534 (1990).
.
Drmanac et al., "An algorithm for the DNA sequence generation from
k-tuple word contents of the minimal number of random fragments" J.
Biomol. Struct. Dyn. 8(5):1085-1102 (1991). .
Drmanac et al, "Sequencing of megabase plus DNA by hybridization:
theory of the method" Genomics 4:114-128 (1989). .
Drmanac et al., "Partial Sequencing by Oligo-hybridization: Concept
and Applications in Genome Analysis" The First Intl. Conf.
Electrophoresis, Supercomputing, and the Human Genome, Eds. Cantor
and Lim, World Scientific, pp. 60-74 (Apr. 10-13, 1990). .
Drmanac et al., "Sequencing by Oligonucleotide Hybridization: A
Promising Framework in Decoding of the Genome Program" The First
Intl. Conf. Electrophoresis, Supercomputing, and the Human Genome,
Eds. Cantor and Lim, World Scientific, pp. 47-59 (Apr. 10-13,
1990). .
Evans et al., "Physical Mapping of Complex Genomes by Cosmid
Multiplex Analysis" Proc. Natl. Acad. Sci. USA 86:5030-5034 (Jul.
1989). .
Feinberg and Vogelstein, "A Technique for Radiolabeling DNA
Restriction Endonuclease Fragments to High Specific Activity" Anal.
Biochem. 137:266-267 (1984) Addendum. .
Fodor et al., "Light-directed, spatially addressable parrallel
chemical synthesis" Science 251:767-773 (1991). .
Hodgson and Fisk, "Hybridization probe size control: optimized
`oligolabeling`" Nucl. Acids Res. 15(15):6295 (1987). .
Khrapko et al., "A method for DNA sequencing by hybridization with
oligonucleotide matrix" DNA Seq. Map 1:375-388 (1991). .
Khrapko et al., "An oligonucleoride hybridization approach to DNA
sequencing" FEBS Lett. 256(1):118-122 (Oct. 1989). .
Lander et al., "Genomic Mapping by Fingerprinting Random Clones: A
Mathematical Analysis" Genomics 2:231-239 (1988). .
Little, P., "Clone maps made simple" Nature 346:611-612(1990).
.
Lysov et al., "Determination of the nucleotide sequence of DNA
using hybridization with oligonucleotides, A new method" Dokl.
Akad. Nauk. SSSR 303:1508-1511 (1988). .
McCray et al., "Properties and uses of photoreactive caged
compounds" Ann. Rev. Biophys. Chem. 18:239-270 (1989). .
Michiels et al., "Molecular Approaches to Genome Analysis: A
Strategy for the Construction of Ordered Overlapping Clone
Libraries" CABIOS 3(3):203-210 (1987). .
Ohtsuka et al., "Studies on transfer ribonucleic acids and related
compounds" Nucl. Acids Res. 1(10):1351-1357 (1974). .
Olson et al., "Random-clone strategy for genomic restriction
mapping in yeast" Proc. Natl. Acad. Sci. USA 83:7826-7830 (Oct.
1986). .
Patchornik et al., "Photosensitive Protecting Group" J. Am. Chem.
Soc. 92(21) :6333-6335 (Oct. 21, 1970). .
Pevzner, P.A., "DNA Physical Mapping and Alternating Eulerian
Cycles in Coloured Graphs" Algorithmica 13(1-2):77-105 (1995).
.
Pevzner, P.A., "i-Tuple DNA sequencing: Computer analysis" J.
Biomol. Struct. Dyn. 7(1):63-73 (1989). .
Pevzner and Waterman, "Multiple Filtration and Approximate Pattern
Matching" Algorithmica 13(1-2):135-154 (1995). .
Pevzner and Waterman, "Generalized Sequence Alignment and Duality"
Adv. Applied Math. 14:139-171 (1993). .
Pfeifer et al., "Genomic sequencing and methylation analysis by
ligation mediated PCR" Science 246:810-813 (Nov. 10, 1989). .
Poustka et al., "Molecular approaches to mammalian genetics" Cold
Spring Harbor Symp. Quant. Biol. 51(Pt. 1):131-139 (1986). .
Sambrook et al., in: Molecular Cloning, A Laboratory Manual, 2nd
Edition, Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY, pp.
11.45-11.47 (1989). .
Seed, B., "Diazotizable arylamine cellulose papers for the coupling
and hybridization of nucleic acids" Nucl. Acids Res.
10(5):1799-1810 (1982). .
Southern et al., "Analyzing and Comparing Nucleic Acid Sequences by
Hybridization to Arrays of Oligonucleotides: Evaluation Using
Experimental Models" Genomics 13:1008-1017 (1992). .
Wood et al., "Base composition-independent hybridization in
tetramethylammonium chloride: a method for oligonucleotide
screening of highly complex gene libraries" Proc. Natl. Acad. Sci.
USA 82:1585-1588 (Mar. 1985)..
|
Primary Examiner: Zitomer; Stephanie W.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of Fodor et al., Ser.
No. 08/168,904, filed Dec. 15, 1993, now abandoned, which is a
continuation of Fodor et al., Ser. No. 07/624,114, filed Dec. 6,
1990, now abandoned, which is a continuation-in-part application of
commonly assigned patent applications Pirrung et al., Ser. No.
07/362,901, filed Jun. 7, 1989, now abandoned; and Pirrung et al.,
Ser. No. 07/492,462, filed Mar. 7, 1990, now U.S. Pat. No.
5,143,854, which are hereby incorporated herein by reference.
Additionally commonly assigned applications Barrett et al., Ser.
No. 07/435,316, filed Nov. 13, 1989, now abandoned; and Barrett et
al., (1993) U.S. Pat. No. 5,252,743 are also incorporated herein by
reference. Additional applications Pirrung et al., Ser. No.
07/624,120, now abandoned, and Dower et al., Ser. No. 07/626,730,
which are also commonly assigned and filed on Dec. 6, 1990 are also
hereby incorporated herein by reference.
Claims
What is claimed is:
1. A method for detecting nucleic acid sequences in two or more
collections of nucleic acid molecules, the method comprising:
(a) providing an array of polynucleotides bound to a solid surface,
each said polynucleotide comprising a determinable nucleic
acid;
(b) contacting the array of polynucleotides with:
(i) a first collection of labelled nucleic acid comprising a
sequence substantially complementary to a nucleic acid of said
array, and
(ii) at least a second collection of labelled nucleic acid
comprising a sequence substantially complementary to a nucleic acid
of said array;
wherein the first and second labels are distinguishable from each
other; and
(c) detecting hybridization of the first and second labelled
complementary nucleic acids to nucleic acids of said arrays.
2. The method of claim 1, wherein the solid support comprises an
array of beads.
3. The method of claim 1, wherein the first and second labels are
fluorescent labels.
4. A method of detecting differential expression of each of a
plurality of genes in a first cell type with respect to expression
of the same genes in a second cell type, said method
comprising:
adding a mixture of labeled nucleic acid from the two cell types to
an array of polynucleotides representing a plurality of known genes
derived from the two cell types, under conditions that result in
hybridization to complementary-sequence polynucleotides in the
array; and
examining the array by fluorescence under fluorescence excitation
conditions in which polynucleotides in the array that are
hybridized to labeled nucleic acid derived from one of the cell
types give a distinct fluorescence emission color and
polynucleotides in the array that are hybridized to labeled nucleic
acid derived from the other cell types give a different
fluorescence emission color.
5. The method of claim 4, wherein the array of polynucleotides is
formed on a substrate with a surface having an array of at least
10.sup.3 distinct polynucleotide in a surface area of about 1
cm.sup.2, each distinct polynucleotide being disposed at a
separate, defined position in said array.
Description
BACKGROUND OF THE INVENTION
The present invention relates to the sequencing, fingerprinting,
and mapping of polymers, particularly biological polymers. The
inventions may be applied, for example, in the sequencing,
fingerprinting, or mapping of nucleic acids, polypeptides,
oligosaccharides, and synthetic polymers.
The relationship between structure and function of macromolecules
is of fundamental importance in the understanding of biological
systems. These relationships are important to understanding, for
example, the functions of enzymes, structural proteins, and
signalling proteins, ways in which cells communicate with each
other, as well as mechanisms of cellular control and metabolic
feedback.
Genetic information is critical in continuation of life processes.
Life is substantially informationally based and its genetic content
controls the growth and reproduction of the organism and its
complements. Polypeptides, which are critical features of all
living systems, are encoded by the genetic material of the cell. In
particular, the properties of enzymes, functional proteins, and
structural proteins are determined by the sequence of amino acids
which make them up. As structure and function are integrally
related, many biological functions may be explained by elucidating
the underlying structural features which provide those functions.
For this reason, it has become very important to determine the
genetic sequences of nucleotides which encode the enzymes,
structural proteins, and other effectors of biological functions.
In addition to segments of nucleotides which encode polypeptides,
there are many nucleotide sequences which are involved in control
and regulation of gene expression.
The human genome project is directed toward determining the
complete sequence of the genome of the human organism. Although
such a sequence would not correspond to the sequence of any
specific individual, it would provide significant information as to
the general organization and specific sequences contained within
segments from particular individuals. It would also provide mapping
information which is very useful for further detailed studies.
However, the need for highly rapid, accurate, and inexpensive
sequencing technology is nowhere more apparent than in a demanding
sequencing project such as this. To complete the sequencing of a
human genome would require the determination of approximately
3.times.10.sup.9, or 3 billion base pairs.
The procedures typically used today for sequencing include the
Sanger dideoxy method, see, e.g., Sanger et al. (1977) Proc. Natl.
Acad. Sci. USA, 74:5463-5467, or the Maxam and Gilbert method, see,
e.g., Maxam et al., (1980) Methods in Enzymology, 65:499-559. The
Sanger method utilizes enzymatic elongation procedures with chain
terminating nucleotides. The Maxam and Gilbert method uses chemical
reactions exhibiting specificity of reaction to generate nucleotide
specific cleavages. Both methods require a practitioner to perform
a large number of complex manual manipulations. These manipulations
usually require isolating homogeneous DNA fragments, elaborate and
tedious preparing of samples, preparing a separating gel, applying
samples to the gel, electrophoresing the samples into this gel,
working up the finished gel, and analyzing the results of the
procedure.
Thus, a less expensive, highly reliable, and labor efficient means
for sequencing biological macromolecules is needed. A substantial
reduction in cost and increase in speed of nucleotide sequencing
would be very much welcomed. In particular, an automated system
would improve the reproducibility and accuracy of procedures. The
present invention satisfies these and other needs.
SUMMARY OF THE INVENTION
The present invention provides improved methods useful for de novo
sequencing of an unknown polymer sequence, for verification of
known sequences, for fingerprinting polymers, and for mapping
homologous segments within a sequence. By reducing the number of
manual manipulations required and automating most of the steps, the
speed, accuracy, and reliability of these procedures are greatly
enhanced.
The production of a substrate having a matrix of positionally
defined regions with attached reagents exhibiting known recognition
specificity can be used for the sequence analysis of a polymer.
Although most directly applicable to sequencing, the present
invention is also applicable to fingerprinting, mapping, and
general screening of specific interactions. The VLSIPS.TM.
Technology (Very Large Scale Immobilized Polymer Synthesis)
substrates will be applied to evaluating other polymers, e.g.,
carbohydrates, polypeptides, hydrocarbon synthetic polymers, and
the like. For these non-polynucleotides, the sequence specific
reagents will usually be antibodies specific for a particular
subunit sequence.
According to one aspect of the masking technique, the invention
provides an ordered method for forming a plurality of polymer
sequences by sequential addition of reagents comprising the step of
serially protecting and deprotecting portions of the plurality, of
polymer sequences for addition of other portions of the polymer
sequences using a binary synthesis strategy.
The present invention also provides a means to automate sequencing
manipulations. The automation of the substrate production method
and of the scan and analysis steps minimizes the need for human
intervention. This simplifies the tasks and promotes
reproducibility.
The present invention provides a composition comprising a plurality
of positionally distinguishable sequence specific reagents attached
to a solid substrate, which reagents are capable of specifically
binding to a predetermined subunit sequence of a preselected
multi-subunit length having at least three subunits, said reagents
representing substantially all possible sequences of said
preselected length. In some embodiments, the subunit sequence is a
polynucleotide or a polypeptide, in others the preselected
multi-subunit length is five subunits and the subunit sequence is a
polynucleotide sequence. In other embodiments, the specific reagent
is an oligonucleotide of at least about five nucleotides.
Alternatively, the specific reagent is a monoclonal antibody.
Usually the specific reagents are all attached to a single solid
substrate, and the reagents comprise about 3000 different
sequences. In other embodiments, the reagents represents at least
about 25% of the possible subsequences of said preselected length.
Usually, the reagents are localized in regions of the substrate
having a density of at least 25 regions per square centimeter, and
often the substrate has a surface area of less than about 4 square
centimeters.
The present invention also provides methods for analyzing a
sequence of a polynucleotide or a polypeptide, said method
comprising the step of:
a) exposing said polynucleotide or polypeptide to a composition as
described.
It also provides useful methods for identifying or comparing a
target sequence with a reference, said method comprising the step
of:
a) exposing said target sequence to a composition as described;
b) determining the pattern of positions of the reagents which
specifically interact with the target sequence; and
c) comparing the pattern with the pattern exhibited by the
reference when exposed to the composition.
The present invention also provides methods for sequencing a
segment of a polynucleotide comprising the steps of:
a) combining:
i) a substrate comprising a plurality of chemically synthesized and
positionally distinguishable oligonucleotides capable of
recognizing defined oligonucleotide sequences; and
ii) a target polynucleotide; thereby forming high fidelity matched
duplex structures of complementary subsequences of known sequence;
and
b) determining which of said reagents have specifically interacted
with subsequences in said target polynucleotide.
In one embodiment, the segment is substantially the entire length
of said polynucleotide.
The invention also provides methods for sequencing a polymer, said
method comprising the steps of:
a) preparing a plurality of reagents which each specifically bind
to a subsequence of preselected length;
b) positionally attaching each of said reagents to one or more
solid phase substrates, thereby producing substrates of
positionally definable sequence specific probes;
c) combining said substrates with a target polymer whose sequence
is to be determined; and
d) determining which of said reagents have specifically interacted
with subsequences in said target polymer.
In one embodiment, the substrates are beads. Preferably, the
plurality of reagents comprise substantially all possible
subsequences of said preselected length found in said target. In
another embodiment, the solid phase substrate is a single substrate
having attached thereto reagents recognizing substantially all
possible subsequences of preselected length found in said
target.
In another embodiment, the method further comprises the step of
analyzing a plurality of said recognized subsequences to assemble a
sequence of said target polymer. In a bead embodiment, at least
some of the plurality of substrates have one subsequence specific
reagent attached thereto, and the substrates are coded to indicate
the sequence specificity of said reagent.
The present invention also embraces a method of using a fluorescent
nucleotide to detect interactions with oligonucleotide probes of
known sequence, said method comprising:
a) attaching said nucleotide to a target unknown polynucleotide
sequence, and
b) exposing said target polynucleotide sequence to a collection of
positionally defined oligonucleotide probes of known sequences to
determine the sequences of said probes which interact with said
target.
In a further refinement, an additional step is included of:
a) collating said known sequences to determine the overlaps of said
known sequences to determine the sequence of said target
sequence.
A method of mapping a plurality of sequences relative to one
another is also provided, the method comprising:
a) preparing a substrate having a plurality of positionally
attached sequence specific probes;
b) exposing each of said sequences to said substrate, thereby
determining the patterns of interaction between said sequence
specific probes and said sequences; and
c) determining the relative locations of said sequence specific
probe interactions on said sequences to determine the overlaps and
order of said sequences.
In one refinement, the sequence specific probes are
oligonucleotides, applicable to where the target sequences are
nucleic acid sequences.
In the nucleic acid sequencing application, the steps of the
sequencing process comprise:
a) producing a matrix substrate having known positionally defined
regions of known sequence specific oligonucleotide probes;
b) hybridizing a target polynucleotide to the positions on the
matrix so that each of the positions which contain oligonucleotide
probes complementary to a sequence on the target hybridize to the
target molecule;
c) detecting which positions have bound the target, thereby
determining sequences which are found on the target; and
d) analyzing the known sequences contained in the target to
determine sequence overlaps and assembling the sequence of the
target therefrom.
The enablement of the sequencing process by hybridization is based
in large part upon the ability to synthesize a large number (e.g.,
to virtually saturate) of the possible overlapping sequence
segments and distinguishing those probes which hybridize with
fidelity from those which have mismatched bases, and to analyze a
highly complex pattern of hybridization results to determine the
overlap regions.
The detecting of the positions which bind the target sequence would
typically be through a fluorescent label on the target. Although a
fluorescent label is probably most convenient, other sorts of
labels, e.g., radioactive, enzyme linked, optically detectable, or
spectroscopic labels may be used. Because the oligonucleotide
probes are positionally defined, the location of the hybridized
duplex will directly translate to the sequences which hybridize.
Thus, analysis of the positions provides a collection of
subsequences found within the target sequence. These subsequences
are matched with respect to their overlaps so as to assemble an
intact target sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates a flow chart for sequence, fingerprint, or
mapping analysis.
FIG. 2 illustrates the process of a VLSIPS.TM. Technology
trinucleotide synthesis.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Overall Description
A. general
B. VLSIPS substrates
C. binary masking
D. applications
E. detection methods and apparatus
F. data analysis
II. Theoretical Analysis
A. simple n-mer structure; theory
B. complications
C. non-polynucleotide embodiments
III. Polynucleotide Sequencing
A. preparation of substrate matrix
B. labeling target polynucleotide
C. hybridization conditions
D. detection; VLSIPS scanning
E. analysis
F. substrate reuse
G. non-polynucleotide aspects
IV. Fingerprinting
A. general
B. preparation of substrate matrix
C. labeling target nucleotides
D. hybridization conditions
E. detection; VLSIPS scanning
F. analysis
G. substrate reuse
H. non-polynucleotide aspects
V. Mapping
A. general
B. preparation of substrate matrix
C. labeling
D. hybridization/specific interaction
E. detection
F. analysis
G. substrate reuse
H. non-polynucleotide aspects
VI. Additional Screening
A. specific interactions
B. sequence comparisons
C. categorizations
D. statistical correlations
VII. Formation of Substrate
A. instrumentation
B. binary masking
C. synthetic methods
D. surface immobilization
VIII. Hybridization/Specific Interaction
A. general
B. important parameters
IX. Detection Methods
A. labeling techniques
B. scanning system
X. Data Analysis
A. general
B. hardware
C. software
XI. Substrate Reuse
A. removal of label
B. storage and preservation
C. processes to avoid degradation of oligomers
XII. Integrated Sequencing Strategy
A. initial mapping strategy
B. selection of smaller clones
C. actual sequencing procedures
XIII. Commercial Applications
A. sequencing
B. fingerprinting
C. mapping
I. OVERALL DESCRIPTION
A. General
The present invention relies in part on the ability to synthesize
or attach specific recognition reagents at known locations on a
substrate, typically a single substrate. In particular, the present
invention provides the ability to prepare a substrate having a very
high density matrix pattern of positionally defined specific
recognition reagents. The reagents are capable of interacting with
their specific targets while attached to the substrate, e.g., solid
phase interactions, and by appropriate labeling of these targets,
the sites of the interactions between the target and the specific
reagents may be derived. Because the reagents are positionally
defined, the sites of the interactions will define the specificity
of each interaction. As a result, a map of the patterns of
interactions with specific reagents on the substrate is convertible
into information on the specific interactions taking place, e.g.,
the recognized features. Where the specific reagents recognize a
large number of possible features, this system allows the
determination of the combination of specific interactions which
exist on the target molecule. Where the number of features is
sufficiently large, the identical same combination, or pattern, of
features is sufficiently unlikely that a particular target molecule
may often be uniquely defined by its features. In the extreme, the
features may actually be the subunit sequence of the target
molecule, and a given target sequence may be uniquely defined by
its combination of features.
In particular, the methodology is applicable to sequencing
polynucleotides. The specific sequence recognition reagents will
typically be oligonucleotide probes which hybridize with
specificity to subsequences found on the target sequence. A
sufficiently large number of those probes allows the fingerprinting
of a target polynucleotide or the relative mapping of a collection
of target polynucleotides, as described in greater detail
below.
In the high resolution fingerprinting provided by a saturating
collection of probes which include all possible subsequences of a
given size, e.g., 10-mers, collating of all the subsequences and
determination of specific overlaps will be derived and the entire
sequence can usually be reconstructed.
Although a polynucleotide sequence analysis is a preferred
embodiment, for which the specific reagents are most easily
accessible, the invention is also applicable to analysis of other
polymers, including polypeptides, carbohydrates, and synthetic
polymers, including .alpha.-, .beta.-, and .omega.-amino acids,
polyurethanes,. polyesters, polycarbonates, polyureas, polyamides,
polyethyleneimines, polyarylene sulfides, polysiloxanes,
polyimides, polyacetates, and mixed polymers. Various optical
isomers, e.g., various D- and L- forms of the monomers, may be
used.
Sequence analysis will take the form of complete sequence
determination, to the level of the sequence of individual subunits
along the entire length of the target sequence. Sequence analysis
also takes the form of sequence homology, e.g., less than absolute
subunit resolution, where "similarity" in the sequence will be
detectable, or the form of selective sequences of homology
interspersed at specific or irregular locations.
In either case, the sequence is determinable at selective
resolution or at particular locations. Thus, the hybridization
method will be useful as a means for identification, e.g., a
"fingerprint", much like a Southern hybridization method is used.
It is also useful to map particular target sequences.
B. VLSIPS.TM. Technology
The invention is enabled by the development of technology to
prepare substrates on which specific reagents may be either
positionally attached or synthesized. In particular, the very large
scale immobilized polymer synthesis (VLSIPS.TM.) technology allows
for the very high density production of an enormous diversity of
reagents mapped out in a known matrix pattern on a substrate. These
reagents specifically recognize subsequences in a target polymer
and bind thereto, producing a map of positionally defined regions
of interaction. These map positions are convertible into actual
features recognized, and thus would be present in the target
molecule of interest.
As indicated, the sequence specific recognition reagents will often
be oligonucleotides which hybridize with fidelity and
discrimination to the target sequence. For use with other polymers,
monoclonal or polyclonal antibodies having high sequence
specificity will often be used.
In the generic sense, the VLSIPS technology allows the production
of a substrate with a high density matrix of positionally mapped
regions with specific recognition reagents attached at each
distinct region. By use of protective groups which can be
positionally removed, or added, the regions can be activated or
deactivated for addition of particular reagents or compounds.
Details of the protection are described below and in related
Pirrung et al. (1992)U.S. Pat. No. 5,143,854. In a preferred
embodiment, photosensitive protecting agents will be used and the
regions of activation or deactivation may be controlled by
electro-optical and optical methods, similar to many of the
processes used in semiconductor wafer and chip fabrication.
In the nucleic acid nucleotide sequencing application, a VLSIPS
substrate is synthesized having positionally defined
oligonucleotide probes. See Pirrung et al. (1992) U.S. Pat. No.
5,143,854; and U.S. Ser. No. 07/624,120, now abandoned. By use of
masking technology and photosensitive synthetic subunits, the
VLSIPS apparatus allows for the stepwise synthesis of polymers
according to a positionally defined matrix pattern. Each
oligonucleotide probe will be synthesized at known and defined
positional locations on the substrate. This forms a matrix pattern
of known relationship between position and specificity of
interaction. The VLSIPS technology allows the production of a very
large number of different oligonucleotide probes to be
simultaneously and automatically synthesized including numbers in
excess of about 10.sup.2, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6,
or even more, and at densities of at least about 10.sup.2, 10.sup.3
/cm.sup.2, 10.sup.4 /cm.sup.2, 10.sup.5 /cm.sup.2 and up to
10.sup.6 /cm.sup.2 or more. This application discloses methods for
synthesizing polymers on a silicon or other suitably derivatized
substrate, methods and chemistry for synthesizing specific types of
biological polymers on those substrates, apparatus for scanning and
detecting whether interaction has occurred at specific locations on
the substrate, and various other technologies related to the use of
a high density very large scale immobilized polymer substrate. In
particular, sequencing, fingerprinting, and mapping applications
are discussed herein in detail, though related technologies are
described in simultaneously filed applications U.S. Ser. No.
07/624,120, now abandoned; and U.S. Ser. No. 07/517,659; Dower et
al. (1995) U.S. Pat. No. 5,427,908, each of which is hereby
incorporated herein by reference.
In other embodiments, antibody probes will be generated which
specifically recognize particular subsequences found on a polymer.
Antibodies would be generated which are specific for recognizing a
three contiguous amino acid sequence, and monoclonal antibodies may
be preferred. Optimally, these antibodies would not recognize any
sequences other than the specific three amino acid stretch desired
and the binding affinity should be insensitive to flanking or
remote sequences found on a target molecule. Likewise, antibodies
specific for particular carbohydrate linkages or sequences will be
generated. A similar approach could be used for preparing specific
reagents which recognize other polymer subunit sequences. These
reagents would typically be site specifically localized to a
substrate matrix pattern where the regions are closely packed.
These reagents could be individually attached at specific sites on
the substrate in a matrix by an automated procedure where the
regions are positionally targeted by some other specific mechanism,
e.g., one which would allow the entire collection of reagents to be
attached to the substrate in a single reaction. Each reagent could
be separately attached to a specific oligonucleotide sequence by an
automated procedure. This would produce a collection of reagents
where, e.g., each monoclonal antibody would have a unique
oligonucleotide sequence attached to it. By virtue of a VLSIPS
substrate which has different complementary oligonucleotides
synthesized on it, each monoclonal antibody would specifically be
bound only at that site on the substrate where the complementary
oligonucleotide has been synthesized. A crosslinking step would fix
the reagent to the substrate. See, e.g., Dattagupta et al. (1985)
U.S. Pat. No. 4,542,102 and (1987) U.S. Pat. No. 4,713,326; and
Chatterjee, M. et al. (1990) J. Am. Chem. Soc. 112:6397-6399, which
are hereby incorporated herein by reference. This allows a high
density positionally specific collection of specific recognition
reagents, e.g., monoclonal antibodies, to be immobilized to a solid
substrate using an automated system.
The regions which define particular reagents will usually be
generated by selective protecting groups which may be activated or
deactivated. Typically the protecting group will be bound to a
monomer subunit or spatial region, and can be spatially affected by
an activator, such as electromagnetic radiation. Examples of
protective groups with utility herein include nitroveratryl
oxycarbonyl (NVOC), nitrobenzyl oxycarbony (NBOC), dimethyl
dimethoxy benzyloxy carbonyl, 5-bromo-7-nitroindolinyl,
O-hydroxy-.alpha.-methyl cinnamoyl, and 2-oxymethylene
anthraquinone. Examples of activators include ion beams, electric
fields, magnetic fields, electron beams, x-ray, and other forms of
electromagnetic radiation.
C. Binary Masking
In fact, the means for producing a substrate useful for these
techniques are explained in Pirrung et al. (1992) U.S. Pat. No.
5,143,854, which is hereby incorporated herein by reference.
However, there are various particular ways to optimize the
synthetic processes. Many of these methods are described in Ser.
No. 07/624,120, now abandoned.
Briefly, the binary synthesis strategy refers to an ordered
strategy for parallel synthesis of diverse polymer sequences by
sequential addition of reagents which may be represented by a
reactant matrix, and a switch matrix, the product of which is a
product matrix. A reactant matrix is a 1.times.n matrix of the
building blocks to be added. The switch matrix is all or a subset
of the binary numbers from 1 to n arranged in columns. In preferred
embodiments, a binary strategy is one in which at least two
successive steps illuminate half of a region of interest on the
substrate. In most preferred embodiments, binary synthesis refers
to a synthesis strategy which also factors a previous addition
step. For example, a strategy in which a switch matrix for a
masking strategy halves regions that were previously illuminated,
illuminating about half of the previously illuminated region and
protecting the remaining half (while also protecting about half of
previously protected regions and illuminating about half of
previously protected regions). It will be recognized that binary
rounds may be interspersed with non-binary rounds and that only a
portion of a substrate may be subjected to a binary scheme, but
will still be considered to be a binary masking scheme within the
definition herein. A binary "masking" strategy is a binary
synthesis which uses light to remove protective groups from
materials for addition of other materials such as nucleotides or
amino acids.
In particular, this procedure provides a simplified and highly
efficient method for saturating all possible sequences of a defined
length polymer. This masking strategy is also particularly useful
in producing all possible oligonucleotide sequence probes of a
given length.
D. Applications
The technology provided by the present invention has very broad
applications. Although described specifically for polynucleotide
sequences, similar sequencing, fingerprinting, mapping, and
screening procedures can be applied to polypeptide, carbohydrate,
or other polymers. In particular, the present invention may be used
to completely sequence a given target sequence to subunit
resolution. This may be for de novo sequencing, or may be used in
conjunction with a second sequencing procedure to provide
independent verification. See, e.g., (1988) Science 242:1245. For
example, a large polynucleotide sequence defined by either the
Maxam and Gilbert technique or by the Sanger technique may be
verified by using the present invention.
In addition, by selection of appropriate probes, a polynucleotide
sequence can be fingerprinted. Fingerprinting is a less detailed
sequence analysis which usually involves the characterization of a
sequence by a combination of defined features. Sequence
fingerprinting is particularly useful because the repertoire of
possible features which can be tested is virtually infinite.
Moreover, the stringency of matching is also variable depending
upon the application. A Southern Blot analysis may be characterized
as a means of simple fingerprint analysis.
Fingerprinting analysis may be performed to the resolution of
specific nucleotides, or may be used to determine homologies, most
commonly for large segments. In particular, an array of
oligonucleotide probes of virtually any workable size may be
positionally localized on a matrix and used to probe a sequence for
either absolute complementary matching, or homology to the desired
level of stringency using selected hybridization conditions.
In addition, the present invention provides means for mapping
analysis of a target sequence or sequences. Mapping will usually
involve the sequential ordering of a plurality of various
sequences, or may involve the localization of a particular sequence
within a plurality of sequences. This may be achieved by
immobilizing particular large segments onto the matrix and probing
with a shorter sequence to determine which of the large sequences
contain that smaller sequence. Alternatively, relatively shorter
probes of known or random sequence may be immobilized to the matrix
and a map of various different target sequences may be determined
from overlaps. Principles of such an approach are described in some
detail by Evans et al. (1989) "Physical Mapping of Complex Genomes
by Cosmid Multiplex Analysis," Proc. Natl. Acad. Sci. USA
86:5030-5034; Michiels et al. (1987) "Molecular Approaches to
Genome Analysis: A Strategy for the Construction of Ordered Overlap
Clone Libraries," CABIOS 3:203-210; Olsen et al. (1986)
"Random-Clone Strategy for Genomic Restriction Mapping in Yeast,"
Proc. Natl. Acad. Sci. USA 83:7826-7830; Craig, et al. (1990)
"Ordering of Cosmid Clones Covering the Herpes Simplex Virus Type I
(HSV-I) Genome: A Test Case for Fingerprinting by Hybridization,"
Nuc. Acids Res. 18:2653-2660; and Coulson, et al. (1986) "Toward a
Physical Map of the Genome of the Nematode Caenorhabditis elegans,"
Proc. Natl. Acad. Sci. USA 83:7821-7825; each of which is hereby
incorporated herein by reference.
Fingerprinting analysis also provides a means of identification. In
addition to its value in apprehension of criminals from whom a
biological sample, e.g., blood, has been collected, fingerprinting
can ensure personal identification for other reasons. For example,
it may be useful for identification of bodies in tragedies such as
fire, flood, and vehicle crashes. In other cases the identification
may be useful in identification of persons suffering from amnesia,
or of missing persons. Other forensics applications include
establishing the identity of a person, e.g., military
identification "dog tags", or may be used in identifying the source
of particular biological samples. Fingerprinting technology is
described, e.g., in Carrano, et al. (1989) "A High-Resolution,
Fluorescence-Based, Semi-automated method for DNA Fingerprinting,"
Genomics 4:129-136, which is hereby incorporated herein by
reference. See, e.g., table I, for nucleic acid applications, and
corresponding applications may be accomplished using
polypeptides.
TABLE I
VLSIPS.TM. Technology in Nucleic Acids
I. Construction of Chips
II. Applications
A. Sequencing
1. Primary sequencing
2. Secondary sequencing (sequence checking)
3. Large scale mapping
4. Fingerprinting
B. Duplex/Triplex formation
1. Antisense
2. Sequence specific function modulation (e.g. promoter
inhibition)
C. Diagnosis
1. Genetic markers
2. Type markers
a. Blood donors
b. Tissue transplants
D. Microbiology
1. Clinical microbiology
2. Food microbiology
III. Instrumentation
A. Chip machines
B. Detection
IV. Software Development
A. Instrumentation software
B. Data reduction software
C. Sequence analysis software
The fingerprinting analysis may be used to perform various types of
genetic screening. For example, a single substrate may be generated
with a plurality of screening probes, allowing for the simultaneous
genetic screening for a large number of genetic markers. Thus,
prenatal or diagnostic screening can be simplified, economized, and
made more generally accessible.
In addition to the sequencing, fingerprinting, and mapping
applications, the present invention also provides means for
determining specificity of interaction with particular sequences.
Many of these applications were described in Ser. No. 07/362,901,
now abandoned, Pirrung et al. (1992) U.S. Pat. No. 5,143,854; Ser.
No. 07/435,316, and Ser. No. 07/612,671.
E. Detection Methods and Apparatus
An appropriate detection method applicable to the selected labeling
method can be selected. Suitable labels include radionucleotides,
enzymes, substrates, cofactors, inhibitors, magnetic particles,
heavy metal atoms, and particularly fluorescers, chemiluminescers,
and spectroscopic labels. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241.
With an appropriate label selected, the detection system best
adapted for high resolution and high sensitivity detection may be
selected. As indicated above, an optically detectable system, e.g.,
fluorescence or chemilumnescence would be preferred. Other
detection systems may be adapted to the purpose, e.g., electron
microscopy, scanning electron microscopy (SEM), scanning tunneling
electron microscopy (STEM), infrared microscopy, atomic force
microscopy (AFM), electrical condutance, and image plate
transfer.
With a detection method selected, an apparatus for scanning the
substrate will be designed. Apparatus, as described in Ser. No.
07/362,901, now abandoned; or Pirrung et al. (1992) U.S. Pat. No.
5,143,854; or Ser. No. 07/624,120, now abandoned, are particularly
appropriate. Design modifications may also be incorporated
therein.
F. Data Analysis
Data is analyzed by processes similar to those described below in
the section describing theoretical analysis. More efficient
algorithms will be mathematically devised, and will usually be
designed to be performed on a computer. Various computer programs
which may more quickly or efficiently make measurement samples and
distinguish signal from noise will also be devised. See,
particularly, Ser. No. 07/624,120, now abandoned.
The initial data resulting from the detection system is an array of
data indicative of fluorescent intensity versus location on the
substrate. The data are typically taken over regions substantially
smaller than the area in which synthesis of a given polymer has
taken place. Merely by way of example, if polymers were synthesized
in squares on the substrate having dimensions of 500 microns by 500
microns, the data may be taken over regions having dimensions of 5
microns by 5 microns. In most preferred embodiments, the regions
over which florescence data are taken across the substrate are less
than about 1/2 the area of the regions in which individual polymers
are synthesized, preferably less than 1/10 the area in which a
single polymer is synthesized, and most preferably less than 1/100
the area in which a single polymer is synthesized. Hence, within
any area in which a given polymer has been synthesized, a large
number of fluorescence data points are collected.
A plot of number of pixels versus intensity for a scan should bear
a rough resemblance to a bell curve, but spurious data are
observed, particularly at higher intensities. Since it is desirable
to use an average of fluorescent intensity over a given synthesis
region in determining relative binding affinity, these spurious
data will tend to undesirably skew the data.
Accordingly, in one embodiment of the invention the data are
corrected for removal of these spurious data points, and an average
of the data points is thereafter utilized in determining relative
binding efficiency. In general the data are fitted to a base curve
and statistical measures are used to remove spurious data.
In an additional analytical tool, various degeneracy reducing
analogues may be incorporated in the hybridization probes. Various
aspects of this strategy are described, e.g., in Macevicz, S.
(1990) PCT publication number WO 90/04652, which is hereby
incorporated herein by reference.
II. THEORETICAL ANALYSIS
The principle of the hybridization sequencing procedure is based,
in part, upon the ability to determine overlaps of short segments.
The VLSIPS technology provides the ability to generate reagents
which will saturate the possible short subsequence recognition
possibilities. The principle is most easily illustrated by using a
binary sequence, such as a sequence of zeros and ones. Once having
illustrated the application to a binary alphabet, the principle may
easily be understood to encompass three letter, four letter, five
or more letter, even 20 letter alphabets. A theoretical treatment
of analysis of subsequence information to reconstruction of a
target sequence is provided, e.e., in Lysov, Yu., et al. (1988)
Doklady Akademi. Nauk. SSR 303:1508-1511; Khrapko K., et al. (1989)
FEBS Letters 256:118-122; Pevzner, P. (1989) J. of Biomolecular
Structure and Dynamics 7:63-69; and Drmanac, R. et al. (1989)
Genomics 4:114-128; each of which is hereby incorporated herein by
reference.
The reagents for recognizing the subsequences will usually be
specific for recognizing a particular polymer subsequence anywhere
within a target polymer. It is preferable that conditions may be
devised which allow absolute discrimination between high fidelity
matching and very low levels of mismatching. The reagent
interaction will preferably exhibit no sensitivity to flanking
sequences, to the subsequence position within the target, or to any
other remote structure within the sequence. For polynucleotide
sequencing, the specific reagents can be oligonucleotide probes;
for polypeptides and carbohydrates, antibodies will be useful
reagents. Antibody reagents should also be useful for other types
of polymers.
A. Simple n-mer Structure: Theory
1. Simple two letter alphabet: example
A simple example is presented below of how a sequence of ten digits
comprising zeros and ones would be sequenceable using short
segments of five digits. For example, consider the sample ten digit
sequence:
A VLSIPS.TM. Technology substrate could be constructed, as
discussed elsewhere, which would have reagents attached in a
defined matrix pattern which specifically recognize each of the
possible five digit sequences of ones and zeros. The number of
possible five digit subsequences is 2.sup.5 =32. The number of
possible different sequences 10 digits long is 2.sup.10 =1,024. The
five contiguous digit subsequences within a ten digit sequence
number six, i.e., positioned at digits 1-5, 2-6, 3-7, 4-8, 5-9, and
6-10. It will be noted that the specific order of the digits in the
sequence is important and that the order is directional, e.g.,
running left to right versus right to left. The first five digit
sequence contained in the target sequence is 10100. The second is
01001, the third is 10011, the fourth is 00111, the fifth is 01110,
and the sixth is 11100.
The VLSIPS.TM. substrate would have a matrix pattern of
positionally attached reagents which recognize each of the
different 5-mer subsequences. Those reagents which recognize each
of the 6 contained 5-mers will bind the target, and a label allows
the positional determination of where the sequence specific
interaction has occurred. By correlation of the position in the
matrix pattern, the corresponding bound subsequences can be
determined.
In the above-mentioned sequence, six different 5-mer sequences
would be determined to be present. They would be: ##STR1##
Any sequence which contains the first five digit sequence, 10100,
already narrows the number of possible sequences (e.g., from 1024
possible sequences) which contain it to less than about 192
possible sequences.
This 192 is derived from the observation that with the subsequence
10100 at the far left of the sequence, in positions 1-5, there are
only 32 possible sequences. Likewise, for that particular
subsequence in positions 2-6, 3-7, 4-8, 5-9, and 6-10. So, to sum
up all of the sequences that could contain 10100, there are 32 for
each position and 6 positions for a total of about 192 possible
sequences. However, some of these 10 digit sequences will have been
counted twice. Thus, by virtue of containing the 10100 subsequence,
the number of possible 10-mer sequences has been decreased from
1024 sequences to less than about 192 sequences.
In this example, not only do we know that the sequence contains
10100, but we also know that it contains the second five character
sequence, 01001. By virtue of knowing that the sequence contains
10100, we can look specifically to determine whether the sequence
contains a subsequence of five characters which contains the four
leftmost digits plus a next digit to the left. For example, we
would look for a sequence of X1010, but we find that there is none.
Thus, we know that the 10100 must be at the left end of the 10-mer.
We would also look to see whether the sequence contains the
rightmost four digits plus a next digit to the right, e.g., 0100X.
We find that the sequence also contains the sequence 01001, and
that X is a 1. Thus, we know at least that our target sequence has
an overlap of 0100 and has the left terminal sequence 101001.
Applying the same procedure to the second 5-mer, we also know that
the sequence must include a sequence of five digits having the
sequence 1001Y where Y must be either 0 or 1. We look through the
fragments and we see that we have a 10011 sequence within our
target, thus Y is also 1. Thus, we would know that our sequence has
a sequence of the first seven being 1010011.
Moving to the next 5-mer, we know that there must be a sequence of
0011Z, where Z must be either 0 or 1. We look at the fragments
produced above and see that the target sequence contains a 00111
subsequence and Z is 1. Thus, we know the sequence must start with
10100111.
The next 5-mer must be of the sequence 0111W where W must be 0 or
1. Again, looking up at the fragments produced, we see that the
target sequence contains a 01110 subsequence, and W is a 0. Thus,
our sequence to this point is 101001110. We know that the last
5-mer must be either 11100 or 11101. Looking above, we see that it
is 11100 and that must be the last of our sequence. Thus, we have
determined that our sequence must have been 1010011100.
However, it will be recognized from the example above with the
sequences provided therein, that the sequence analysis can start
with any known positive probe subsequence. The determination may be
performed by moving linearly along the sequence checking the known
sequence with a limited number of next positions. Given this
possibility, the sequence may be determined, besides by scanning
all possible oligonucleotide probe positions, by specifically
looking only where the next possible positions would be. This may
increase the complexity of the scanning but may provide a longer
time span dedicated towards scanning and detecting specific
positions of interest relative to other sequence possibilities.
Thus, the scanning apparatus could be set up to work its way along
a sequence from a given contained oligonucleotide to only look at
those positions on the substrate which are expected to have a
positive signal.
It is seen that given a sequence, it can be deconstructed into
n-mers to produce a set of internal contiguous subsequences. From
any given target sequence, we would be able to determine what
fragments would result. The hybridization sequence method depends,
in part, upon being able to work in the reverse, from a set of
fragments of known sequences to the full sequence. In simple cases,
one is able to start at a single position and work in either or
both directions towards the ends of the sequence as illustrated in
the example.
The number of possible sequences of a given length increases very
quickly with the length of that sequence. Thus, a 10-mer of zeros
and ones has 1024 possibilities, a 12-mer has 4096. A 20-mer has
over a million possibilities, and a 30-mer has over a billion.
However, a given 30-mer has, at most, 26 different internal 5-mer
sequences. Thus, a 30 character target sequence having over a
million possible sequences can be substantially defined by only 26
different 5-mers. It will be recognized that the probe
oligonucleotides will preferably, but need not necessarily, be of
identical length, and that the probe sequences need not necessarily
be contiguous in that the overlapping subsequences need not differ
by only a single subunit. Moreover, each position of the matrix
pattern need not be homogeneous, but may actually contain a
plurality of probes of known sequence. In addition, although all of
the possible subsequence specifications would be preferred, a less
than full set of sequences specifications could be used. In
particular, although a substantial fraction will preferably be at
least about 70%, it may be less than that. About 20% would be
preferred, more preferably at least about 30% would be desired.
Higher percentages would be especially preferred.
2. Example of four letter alphabet
A four letter alphabet may be conceptualized in at least two
different ways from the two letter alphabet. One way is to consider
the four possible values at each position and to analogize in a
similar fashion to the binary example each of the overlaps. A
second way is to group the binary digits into groups.
Using the first means, the overlap comparisons are performed with a
four letter alphabet rather than a two letter alphabet. Then, in
contrast to the binary system with 10 positions where 2.sup.10
=1024 possible sequences, in a 4-character alphabet with 10
positions, there will actually be 4.sup.10 =1,048,576 possible
sequences. Thus, the complexity of a four character sequence has a
much larger number of possible sequences compared to a two
character sequence. Note, however, that there are still only 6
different internal 5-mers. For simplicity, we shall examine a 5
character string with 3 character subsequences. Instead of only 1
and 0, the characters may be designated, e.g., A, C, G, and T. Let
us take the sequence GGCTA. The 3-mer subsequences are: ##STR2##
Given these subsequences, there is one sequence, or at most only a
few sequences which would produce that combination of subsequences,
i.e., GGCTA.
Alternatively, with a four character universe, the binary system
can be looked at in pairs of digits. The pairs would be 00, 01, 10,
and 11. In this manner, the earlier used sequence 1010011100 is
looked at as 10,10,01,11,00. Then the first character of two digits
is selected from the possible universe of the four representations
00, 01, 10, and 11. Then a probe would be in an even number of
digits, e.g., not five digits, but, three pairs of digits or six
digits. A similar comparison is performed and the possible overlaps
determined. The 3-pair subsequences are: ##STR3## and the overlap
reconstruction produces 10,10,01,11,00.
The latter of the two conceptual views of the 4 letter alphabet
provides a representation which is similar to what would be
provided in a digital computer. The applicability to a four
nucleotide alphabet is easily seen by assigning, e.g., 00 to A, 01
to C, 10 to G, and 11 to T. And, in fact, if such a correspondence
is used, both examples for the 4 character sequences can be seen to
represent the same target sequence. The applicability of the
hybridization method and its analysis for determining the ultimate
sequence is easily seen if A is the representation of adenine, C is
the representation of cytosine, G is the representation of guanine,
and T is the representation of thymine or uracil.
3. Generalization to m-letter Alphabet
This reconstruction process may be applied to polymers of virtually
any number of possible characters in the alphabet, and for
virtually any length sequence to be sequenced, though limitations,
as discussed below, will limit its efficiency at various extremes
of length. It will be recognized that the theory can be applied to
a large diversity of systems where sequence is important.
For example, the method could be applied to sequencing of a
polypeptide. A polypeptide can have any of twenty natural amino
acid possibilities at each position. A twenty letter alphabet is
amenable to sequencing by this method so long as reagents exist for
recognizing shorter subsequences therein. A preferred reagent for
achieving that goal would be a set of monoclonal antibodies each of
which recognizes a specific three contiguous amino acid
subsequence. A complete set of antibodies which recognize all
possible subsequences of a given length, e.g., 3 amino acids, and
preferably with a uniform affinity, would be 20.sup.3 =8000
reagents.
It will also be recognized that each target sequence which is
recognized by the specific reagents need not have homogeneous
termini. Thus, fragments of the entire target sequence will also be
useful for hybridizing appropriate subsequences. It is, however,
preferable that there not be a significant amount of labeled
homogeneous contaminating extraneous sequences. This constraint
does usually require the purification of the target molecule to be
sequenced, but a specific label technique would dispense with a
purification requirement if the unlabeled extraneous sequences do
not interfere with the labeled sequences.
In addition, conformational effects of target polypeptide folding
may, in certain embodiments, be negligible if the polypeptide is
fragmented into sufficiently small peptides, or if the interaction
is performed under conditions where conformation, but not specific
interaction, is disrupted.
B. Complications
Two obvious complications exist with the method of sequence
analysis by hybridization. The first results from a probe of
inappropriate length while the second relates to internally
repeated sequences.
The first obvious complication is a problem which arises from an
inappropriate length of recognition sequence, which causes problems
with the specificity of recognition. For example, if the recognized
sequence is too short, every sequence which is utilized will be
recognized by every probe sequence. This occurs, e.g., in a binary
system where the probes are each of sequences which occur
relatively frequently, e.g., a two character probe for the binary
system. Each possible two character probe would be expected to
appear 1/4 of the time in every single two character position.
Thus, the above sequence example would be recognized by each of the
00, 10, 01, and 11. Thus, the sequence information is virtually
lost because the resolution is too low and each recognition reagent
specifically binds at multiple sites on the target sequence.
The number of different probes which bind to a target depends on
the relationship between the probe length and the target length. At
the extreme of short probe length, the just mentioned problem
exists of excessive redundancy and lack of resolution. The lack of
stability in recognition will also be a problem with extremely
short probes. At the extreme of long probe length, each entire
probe sequence is on a different position of a substrate. However,
a problem arises from the number of possible sequences, which goes
up dramatically with the length of the sequence. Also, the
specificity of recognition begins to decrease as the contribution
to binding by any particular subunit may become sufficiently low
that the system fails to distinguish the fidelity of recognition.
Mismatched hybridization may be a problem with the polynucleotide
sequencing applications, though the fingerprinting and mapping
applications may not be so strict in their fidelity requirements.
As indicated above, a thirty position binary sequence has over a
million possible sequences, a number which starts to become
unreasonably large in its required number of different sequences,
even though the target length is still very short. Preparing a
substrate with all sequence possibilities for a long target may be
extremely difficult due to the many different oligomers which must
be synthesized.
The above example illustrates how a long target sequence may be
reconstructed with a reasonably small number of shorter
subsequences. Since the present day resolution of the regions of
the substrate having defined oligomer probes attached to the
substrate approaches about 10 microns by 10 microns for resolvable
regions, about 10.sup.6, or 1 million, positions can be placed on a
one centimeter square substrate. However, high resolution systems
may have particular disadvantages which may be outweighed using the
lower density substrate matrix pattern. For this reason, a
sufficiently large number of probe sequences can be utilized so
that any given target sequence may be determined by hybridization
to a relatively small number of probes.
A second complication relates to convergence of sequences to a
single subsequence. This will occur when a particular subsequence
is repeated in the target sequence. This problem can be addressed
in at least two different ways. The first, and simpler way, is to
separate the repeat sequences onto two different targets. Thus,
each single target will not have the repeated sequence and can be
analyzed to its end. This solution, however, complicates the
analysis by requiring that some means for cutting at a site between
the repeats can be located. Typically a careful sequencer would
want to have two intermediate cut points so that the intermediate
region can also be sequenced in both directions across each of the
cut points. This problem is inherent in the hybridization method
for sequencing but can be minimized by using a longer known probe
sequence so that the frequency of probe repeats is decreased.
Knowing the sequence of flanking sequences of the repeat will
simplify the use of polymerase chain reaction (PCR) or a similar
technique to further definitively determine the sequence between
sequence repeats. Probes can be made to hybridize to those known
sequences adjacent the repeat sequences, thereby producing new
target sequences for analysis.
See, e.g., Innis et al. (eds.) (1990) PCR Protocols: A Guide to
Methods and Applications, Academic Press; and methods for synthesis
of oligonucleotide probes, see, e.g., Gait (1984) Oligonucleotide
Synthesis: A Practical Approach, IRL Press, Oxford.
Other means for dealing with convergence problems include using
particular longer probes, and using degeneracy reducing analogues,
see, e.g., Macevicz, S. (1990) PCT publication number WO 90/04652,
which is hereby incorporated herein by reference. By use of
stretches of the degeneracy reducing analogues with other probes in
particular combinations, the number of probes necessary to fully
saturate the possible oligomer probes is decreased. For example,
with a stretch of 12-mers having the central 4-mer of degenerate
nucleotides, in combination with all of the possible 8-mers, the
collection numbers twice the number of possible 8-mers, e.g.
65,536+65,536=131,072, but the population provides screening
equivalent to all possible 12-mers.
By way of further explanation, all possible oligonucleotide 8-mers
may be depicted in the fashion:
in which there are 4.sup.8 =65,536 possible 8-mers. As described in
Ser. No. 07/624,120; now abandoned, producing all possible 8-mers
requires 4.times.8=32 chemical binary synthesis steps to produce
the entire matrix pattern of 65,536 8-mer possibilities. By
incorporating degeneracy reducing nucleotides, D's, which hybridize
nonselectively to any corresponding complementary nucleotide, new
oligonucleotides 12-mers can be made in the fashion:
in which there are again, as above, only 4.sup.8 =65,536 possible
"12-mers", which in reality only have 8 different nucleotides.
However, it can be seen that each possible 12-mer probe could be
represented by a group of the two 8-mer types. Moreover, repeats of
less than 12 nucleotides would not converge, or cause repeat
problems in the analysis. Thus, instead of requiring a collection
of probes corresponding to all 12-mers, or 4.sup.12 =16,777,216
different 12-mers, the same information can be derived by making 2
sets of "8-mers" consisting of the typical 8-mer collection of
4.sup.8 =65,536 and the "12-mer" set with the degeneracy reducing
analogues, also requiring making 4.sup.8 =65,536. The combination
of the two sets, requires making 65,536+65,536=131,072 different
molecules, but giving the information of 16,777,216 molecules.
Thus, incorporating the degeneracy reducing analogue decreases the
number of molecules necessary to get 12-mer resolution by a factor
of about 128-fold.
C. Non-polynucleotide Embodiments
The above example is directed towards a polynucleotide embodiment.
This application is relatively easily achieved because the specific
reagents will typically be complementary oligonucleotides, although
in certain embodiments other specific reagents may be desired. For
example, there may be circumstances where other than complementary
base pairing will be utilized. The polynucleotide targets, will
usually be single strand, but may be double or triple stranded in
various applications. However, a triple stranded specific
interaction might be sometimes desired, or a protein or other
specific binding molecule may be utilized. For example, various
promoter or DNA sequence specific binding proteins might be used,
including, e.g., restriction enzyme binding domains, other binding
domains, and antibodies. Thus, specific recognition reagents
besides oligonucleotides may be utilized.
For other polymer targets, the specific reagents will often be
polypeptides. These polypeptides may be protein binding domains
from enzymes or other proteins which display specificity for
binding. Usually an antibody molecule may be used, and monoclonal
antibodies may be particularly desired. Classical methods may be
applied for preparing antibodies, see, e.g., Harlow and Lane (1988)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, New York;
and Goding (1986) Monoclonal Antibodies: Principles and Practice
(2d Ed.) Academic Press, San Diego. Other suitable techniques for
in vitro exposure of lymphocytes to the antigens or selection of
libraries of antibody binding sites are described, e.g., in Huse et
al. (1989) Science 246:1275-1281; and Ward et al. 91989) Nature
341:544-546, each of which is hereby incorporated herein by
reference. Unusual antibody production methods are also described,
e.g., in Hendricks et al. (1989) BioTechnology 7:1271-1274; and
Hiatt et al. (1989) Nature 342:76-78, each of which is hereby
incorporated herein by reference. Other molecules which may exhibit
specific binding interaction may be useful for attachment to a
VLSIPS substrate by various methods, including the caged biotin
methods, see, e.g., Ser. No. 07/435,316, now abandoned, and Barrett
et al. (1993) U.S. Pat. No. 5,252,743.
The antibody specific reagents should be particularly useful for
the polypeptide, carbohydrate, and synthetic polymer applications.
Individual specific reagents might be generated by an automated
process to generate the number of reagents necessary to
advantageously use the high density positional matrix pattern. In
an alternative approach, a plurality of hybridoma cells may be
screened for their ability to bind to a VLSIPS matrix possessing
the desired sequences whose binding specificity is desired. Each
cell might be individually grown up and its binding specificity
determined by VLSIPS apparatus and technology. An alternative
strategy would be to expose the same VLSIPS matrix to a polyclonal
serum of high titer. By a successively large volume of serum and
different animals, each region of the VLSIPS substrate would have
attached to it a substantial number of antibody molecules with
specificity of binding. The substrate, with non-covalently bound
antibodies could be derivatized and the antibodies transferred to
an adjacent second substrate in the matrix pattern in which the
antibody molecules had attached to the first matrix. If the
sensitivity of detection of binding interaction is sufficiently
high, such a low efficiency transfer of antibody molecules may
produce a sufficiently high signal to be useful for many purposes,
including the sequencing applications.
In another embodiment, capillary forces may be used to transfer the
selected reagents to a new matrix, to which the reagents would be
positionally attached in the pattern of the recognized sequences.
Or, the reagents could be transversely electrophoresed,
magnetically transferred, or otherwise transported to a new
substrate in their retained positional pattern.
III. POLYNUCLEOTIDE SEQUENCING
In principle, the making of a substrate having a positionally
defined matrix pattern of all possible oligonucleotides of a given
length involves a conceptually simple method of synthesizing each
and every different possible oligonucleotide, and affixing them to
a definable position. Oligonucleotide synthesis is presently
mechanized and enabled by current technology, see, e.g., Ser. No.
07/362,901, now abandoned; Pirrung et al. (1992) U.S. Pat. No.
5,143,854; and instruments supplied by Applied Biosystems, Foster
City, Calif.
A. Preparation of Substrate Matrix
The production of the collection of specific oligonucleotides used
in polynucleotide sequencing may be produced in at least two
different ways. Present technology certainly allows production of
ten nucleotide oligomers on a solid phase or other synthesizing
system. See, e.g., instrumentation provided by Applied Biosystems,
Foster City, Calif. Although a single oligonucleotide can be
relatively easily made, a large collection of them would typically
require a fairly large amount of time and investment. For example,
there are 4.sup.10 =1,048,576 possible ten nucleotide oligomers.
Present technology allows making each and every one of them in a
separate purified form though such might be costly and
laborious.
Once the desired repertoire of possible oligomer sequences of a
given length have been synthesized, this collection of reagents may
be individually positionally attached to a substrate, thereby
allowing a batchwise hybridization step. Present technology also
would allow the possibility of attaching each and every one of
these 10-mers to a separate specific position on a solid matrix.
This attachment could be automated in any of a number of ways,
particularly through the use of a caged biotin type linking. This
would produce a matrix having each of different possible
10-mers.
A batchwise hybridization is much preferred because of its
reproducibility and simplicity. An automated process of attaching
various reagents to positionally defined sites on a substrate is
provided in Pirrung et al. (1992) U.S. Pat. No. 5,143,854; Ser. No.
07/624,120, now abandoned; and Barrett et al. (1993) U.S. Pat. No.
5,252,743; each of which is hereby incorporated herein by
reference.
Instead of separate synthesis of each oligonucleotide, these
oligonucleotides are conveniently synthesized in parallel by
sequential synthetic processes on a defined matrix pattern as
provided in Pirrung et al. (1992) U.S. Pat. No. 5,143,854; and Ser.
No. 07/624,120, now abandoned, which are incorporated herein by
reference. Here, the oligonucleotides are synthesized stepwise on a
substrate at positionally separate and defined positions. Use of
photosensitive blocking reagents allows for defined sequences of
synthetic steps over the surface of a matrix pattern. By use of the
binary masking strategy, the surface of the substrate can be
positioned to generate a desired pattern of regions, each having a
defined sequence oligonucleotide synthesized and immobilized
thereto.
Although the prior art technology can be used to generate the
desired repertoire of oligonucleotide probes, an efficient and cost
effective means would be to use the VLSIPS technology described in
Pirrung et al. (1992) U.S. Pat. No. 5,143,854 and Ser. No.
07/624,120, now abandoned. In this embodiment, the photosensitive
reagents involved in the production of such a matrix are described
below.
The regions for synthesis may be very small, usually less than
about 100 .mu.m.times.100 .mu.m, more usually less than about 50
.mu.m.times.50 .mu.m. The photolithography technology allows
synthetic regions of less than about 10 .mu.m.times.10 .mu.m, about
3 .mu.m.times.3 .mu.m, or less. The detection also may detect such
sized regions, though larger areas are more easily and reliably
measured.
At a size of about 30 microns by 30 microns, one million regions
would take about 11 centimeters square or a single wafer of about 4
centimeters by 4 centimeters. Thus the present technology provides
for making a single matrix of that size having all one million plus
possible oligonucleotides. Region size is sufficiently small to
correspond to densities of at least about 5 regions/cm.sup.2, 20
regions/cm.sup.2, 50 regions/cm.sup.2. 100 regions/cm.sup.2, and
greater, including 300 regions/cm.sup.2, 1000 regions/cm.sup.2, 3K
regions/cm.sup.2, 10K regions/cm.sup.2, 30K regions/cm.sup.2, 100K
regions/cm.sup.2, 300K regions/cm.sup.2 or more, even in excess of
one million regions/cm.sup.2.
Although the pattern of the regions which contain specific
sequences is theoretically not important, for practical reasons
certain patterns will be preferred in synthesizing the
oligonucleotides. The application of binary masking algorithms for
generating the pattern of known oligonucleotide probes is described
in related Ser. No. 07/624,120, now abandoned, which was filed
simultaneously with this application. By use of these binary masks,
a highly efficient means is provided for producing the substrate
with the desired matrix pattern of different sequences. Although
the binary masking strategy allows for the synthesis of all lengths
of polymers, the strategy may be easily modified to provide only
polymers of a given length. This is achieved by omitting steps
where a subunit is not attached.
The strategy for generating a specific pattern may take any of a
number of different approaches. These approaches are well described
in related application Ser. No. 07/624,120, now abandoned, and
include a number of binary masking approaches which will not be
exhaustively discussed herein. However, the binary masking and
binary synthesis approaches provide a maximum of diversity with a
minimum number of actual synthetic steps.
The length of oligonucleotides used in sequencing applications will
be selected on criteria determined to some extent by the practical
limits discussed above. For example, if probes are made as
oligonucleotides, there will be 65,536 possible eight nucleotide
sequences. If a nine subunit oligonucleotide is selected, there are
262,144 possible permeations of sequences. If a ten-mer
oligonucleotide is selected, there are 1,048,576 possible
permeations of sequences. As the number gets larger, the required
number of positionally defined subunits necessary to saturate the
possibilities also increases. With respect to hybridization
conditions, the length of the matching necessary to confer
stability of the conditions selected can be compensated for. See,
e.g., Kanehisa, M. (1984) Nuc. Acids Res. 12:203-213, which is
hereby incorporated herein by reference.
Although not described in detail here, but below for
oligonucleotide probes, the VLSIPS technology would typically use a
photosensitive protective group on an oligonucleotide. Sample
oligonucleotides are shown in FIG. 1. In particular, the
photoprotective group on the nucleotide molecules may be selected
from a wide variety of positive light reactive groups preferably
including nitro aromatic compounds such as o-nitrobenzyl
derivatives or benzylsulfonyl. See, e.g., Gait (1984)
Oligonucleotide Synthesis: A Practical Approach, IRL Press, Oxford,
which is hereby incorporated herein by reference. In a preferred
embodiment, 6-nitro-veratryl oxycarbony (NVOC), 2-nitrobenzyl
oxycarbonyl (NBOC), or .alpha.,.alpha.-dimethyl-dimethoxybenzyl
oxycarbonyl (DEZ) is used. Photoremovable protective groups are
described in, e.g., Patchornik (1970) J. Amer. Chem. Soc.
92:6333-6335; and Amit et al. (1974) J. Organic Chem. 39:192-196;
each of which is hereby incorporated herein by reference.
A preferred linker for attaching the oligonucleotide to a silicon
matrix is illustrated in FIG. 2. A more detailed description is
provided below. A photosensitive blocked nucleotide may be attached
to specific locations of unblocked prior cycles of attachments on
the substrate and can be successively built up to the correct
length oligonucleotide probe.
It should be noted that multiple substrates may be simultaneously
exposed to a single target sequence where each substrate is a
duplicate of one another or where, in combination, multiple
substrates together provide the complete or desired subset of
possible subsequences. This provides the opportunity to overcome a
limitation of the density of positions on a single substrate by
using multiple substrates. In the extreme case, each probe might be
attached to a single bead or substrate and the beads sorted by
whether there is a binding interaction. Those beads which do bind
might be encoded to indicate the subsequence specificity of
reagents attached thereto.
Then, the target may be bound to the whole collection of beads and
those beads that have appropriate specific reagents on them will
bind to the target. Then a sorting system may be utilized to sort
those beads that actually bind the target from those that do not.
This may be accomplished by presently available cell sorting
devices or a similar apparatus. After the relatively small number
of beads which have bound the target have been collected, the
encoding scheme may be read off to determine the specificity of the
reagent on the bead. An encoding system may include a magnetic
system, a shape encoding system, a color encoding system, or a
combination of any of these, or any other encoding system. Once
again, with the collection of specific interactions that have
occurred, the binding may be analyzed for sequence information,
fingerprint information, or mapping information.
The parameters of polynucleotide sizes of both the probes and
target sequences are determined by the applications and other
circumstances. The length of the oligonucleotide probes used will
depend in part upon the limitations of the VLSIPS technology to
provide the number of desired probes. For example, in an absolute
sequencing application, it is often useful to have virtually all of
the possible oligonucleotides of a given length. As indicated
above, there are 65,536 8-mers, 262,144 9-mers, 1,048,576 10-mers,
4,194,304 11-mers, etc. As the length of the oligomer increases the
number of different probes which must be synthesized also increases
at a rate of a factor of 4 for every additional nucleotide.
Eventually the size of the matrix and the limitations in the
resolution of regions in the matrix will reach the point where an
increase in number of probes becomes disadvantageous. However, this
sequencing procedure requires that the system be able to
distinguish, by appropriate selection of hybridization and washing
conditions, between binding of absolute fidelity and binding of
complementary sequences containing mismatches. On the other hand,
if the fidelity is unnecessary, this discrimination is also
unnecessary and a significantly longer probe may be used.
Significantly longer probes would typically be useful in
fingerprinting or mapping applications.
The length of the probe is selected for a length that will allow
the probe to bind with specificity to possible targets. The
hybridization conditions are also very important in that they will
determine how closely the homology of complementary binding will be
detected. In fact, a single target may be evaluated at a number of
different conditions to determine its spectrum of specificity for
binding particular probes. This may find use in a number of other
applications besides the polynucleotide sequencing fingerprinting
or mapping. For example, it will be desired to determine the
spectrum of binding affinities and specificities of cell surface
antigens with binding by particular antibodies immobilized on the
substrate surface, particularly under different interaction
conditions. In a related fashion, different regions with reagents
having differing affinities or levels of specificity may allow such
a spectrum to be defined using a single incubation, where various
regions, at a given hybridization condition, show the binding
affinity. For example, fingerprint probes of various lengths, or
with specific defined non-matches may be used. Unnatural
nucleotides or nucleotides exhibiting modified specificity of
complementary binding are described in greater detail in Macevicz
(1990) PCT pub. No. WO 90/04652; and see the section on modified
nucleotides in the Sigma Chemical Company catalogue.
B. Labeling Target Nucleotide
The label used to detect the target sequences will be determined,
in part, by the detection methods being applied. Thus, the labeling
method and label used are selected in combination with the actual
detecting systems being used.
Once a particular label has been selected, appropriate labeling
protocols will be applied, as described below for specific
embodiments. Standard labeling protocols for nucleic acids are
described, e.g., in Sambrook et al.; Kambara, H. et al. (1988)
BioTechnology 6:816-821; Smith, L. et al. (1985) Nuc. Acids Res.
13:2399-2412; for polypeptides, see, e.g., Allen G. (1989)
Sequencing of Proteins and Peptides, Elsevier, New York, especially
chapter 5, and Greenstein and Winitz (1961) Chemistry of the Amino
Acids, Wiley and Sons, New York. Carbohydrate labeling is
described, e.g., in Chaplin and Kennedy (1986) Carbohydrate
Analysis: A Practical Approach, IRL Press, Oxford. Labeling of
other polymers will be performed by methods applicable to them as
recognized by a person having ordinary skill in manipulating the
corresponding polymer.
In some embodiments, the target need not actually be labeled if a
means for detecting where interaction takes place is available. As
described below, for a nucleic acid embodiment, such may be
provided by an intercalating dye which intercalates only into
double stranded segments, e.g., where interaction occurs. See,
e.g., Sheldon et al. U.S. Pat. No. 4,582,789.
In many uses, the target sequence will be absolutely homogeneous,
both with respect to the total sequence and with respect to the
ends of each molecule. Homogeneity with respect to sequence is
important to avoid ambiguity. It is preferable that the target
sequences of interest not be contaminated with a significant amount
of labeled contaminating sequences. The extent of allowable
contamination will depend on the sensitivity of the detection
system and the inherent signal to noise of the system. Homogeneous
contamination sequences will be particularly disruptive of the
sequencing procedure.
However, although the target polynucleotide must have a unique
sequence, the target molecules need not have identical ends. In
fact, the homogeneous target molecule preparation may be randomly
sheared to increase the numerical number of molecules. Since the
total information content remains the same, the shearing results
only in a higher number of distinct sequences which may be labeled
and bind to the probe. This fragmentation may give a vastly
superior signal relative to a preparation of the target molecules
having homogeneous ends. The signal for the hybridization is likely
to be dependent on the numerical frequency of the target-probe
interactions. If a sequence is individually found on a larger
number of separate molecules a better signal will result. In fact,
shearing a homogeneous preparation of the target may often be
preferred before the labeling procedure is performed, thereby
producing a large number of labeling groups associated with each
subsequence.
C. Hybridization Conditions
The hybridization conditions between probe and target should be
selected such that the specific recognition interaction, i.e.,
hybridization, of the two molecules is both sufficiently specific
and sufficiently stable. See, e.g., Hames and Higgins (1985)
Nucleic Acid Hybridisation: A Practical Approach, IRL Press,
Oxford. These conditions will be dependent both on the specific
sequence and often on the guanine and cytosine (GC) content of the
complementary hybrid strands. The conditions may often be selected
to be universally equally stable independent of the specific
sequences involved. This typically will make use of a reagent such
as an alkylammonium buffer. See, Wood et al. (1985) "Base
Composition-independent Hybridization in Tetramethylammonium
Chloride: A Method for Oligonucleotide Screening of Highly Complex
Gene Libraries," Proc. Natl. Acad. Sci. USA, 82:1585-1588; and
Krupov et al. (1989) "An Oligonucleotide Hybridization Approach to
DNA Sequencing," FEBS Letters, 256:118-122; each of which is hereby
incorporated herein by reference. An alkylammonium buffer tends
to-minimize differences in hybridization rate and stability due to
GC content. By virtue of the fact that sequences then hybridize
with approximately equal affinity and stability, there is
relatively little bias in strength or kinetics of binding for
particular sequences. Temperature and salt conditions along with
other buffer parameters should be selected such that the kinetics
of renaturation should be essentially independent of the specific
target subsequence or oligonucleotide probe involved. In order to
ensure this, the hybridization reactions will usually be performed
in a single incubation of all the substrate matrices together
exposed to the identical same target probe solution under the same
conditions.
Alternatively, various substrates may be individually treated
differently. Different substrates may be produced, each having
reagents which bind to target subsequences with substantially
identical stabilities and kinetics of hybridization. For example,
all of the high GC content probes could be synthesized on a single
substrate which is treated accordingly. In this embodiment, the
arylammonium buffers could be unnecessary. Each substrate is then
treated in a manner such that the collection of substrates show
essentially uniform binding and the hybridization data of target
binding to the individual substrate matrix is combined with the
data from other substrates to derive the necessary subsequence
binding information. The hybridization conditions will usually be
selected to be sufficiently specific such that the fidelity of base
matching will be properly discriminated. Of course, control
hybridizations should be included to determine the stringency and
kinetics of hybridization.
D. Detection; VLSIPS.TM. Technology Scanning
The next step of the sequencing process by hybridization involves
labeling of target polynucleotide molecules. A quickly and easily
detectable signal is preferred. The VLSIPS.TM. Technology apparatus
is designed to easily detect a fluorescent label, so fluorescent
tagging of the target sequence is preferred. Other suitable labels
include heavy metal labels, magnetic probes, chromogenic labels
(e.g., phosphorescent labels, dyes, and fluorophores) spectroscopic
labels, enzyme linked labels, radioactive labels, and labeled
binding proteins. Additional labels are described in U.S. Pat. No.
4,366,241, which is incorporated herein by reference.
The detection methods used to determine where hybridization has
taken place will typically depend upon the label selected above.
Thus, for a fluorescent label a fluorescent detection step will
typically be used. Pirrung et al. (1992) U.S. Pat. No. 5,143,854
and Ser. No. 07/624,120, now abandoned, describe apparatus and
mechanisms for scanning a substrate matrix using fluorescence
detection, but a similar apparatus is adaptable for other optically
detectable labels.
The detection method provides a positional localization of the
region where hybridization has taken place. However, the position
is correlated with the specific sequence of the probe since the
probe has specifically been attached or synthesized at a defined
substrate matrix position. Having collected all of the data
indicating the subsequences present in the target sequence, this
data may be aligned by overlap to reconstruct the entire sequence
of the target, as illustrated above.
It is also possible to dispense with actual labeling if some means
for detecting the positions of interaction between the sequence
specific reagent and the target molecule are available. This may
take the form of an additional reagent which can indicate the sites
either of interaction, or the sites of lack of interaction, e.g., a
negative label. For the nucleic acid embodiments, locations of
double strand interaction may be detected by the incorporation of
intercalating dyes, or other reagents such as antibody or other
reagents that recognize helix formation, see, e.g., Sheldon, et al.
(1986) U.S. Pat. No. 4,582,789, which is hereby incorporated herein
by reference.
E. Analysis
Although the reconstruction can be performed manually as
illustrated above, a computer program will typically be used to
perform the overlap analysis. A program may be written and run on
any of a large number of different computer hardware systems. The
variety of operating systems and languages useable will be
recognized by a computer software engineer. Various different
languages may be used, e.g., BASIC; C; PASCAL; etc. A simple flow
chart of data analysis is illustrated in FIG. 2.
F. Substrate Reuse
Finally, after a particular sequence has been hybridized and the
pattern of hybridization analyzed, the matrix substrate should be
reusable and readily prepared for exposure to a second or
subsequent target polynucleotides. In order to do so, the hybrid
duplexes are disrupted and the matrix treated in a way which
removes all traces of the original target. The matrix may be
treated with various detergents or solvents to which the substrate,
the oligonucleotide probes, and the linkages to the substrate are
inert. This treatment may include an elevated temperature
treatment, treatment with organic or inorganic solvents,.
modifications in pH, and other means for disrupting specific
interaction. Thereafter, a second target may actually be applied to
the recycled matrix and analyzed as before.
G. Non-Polynucleotide Aspects
Although the sequencing, fingerprinting, and mapping functions will
make use of the natural sequence recognition property of
complementary nucleotide sequences, the non-polynucleotide
sequences typically require other sequence recognition reagents.
These reagents will take the form, typically, of proteins
exhibiting binding specificity, e.g., enzyme binding sites or
antibody binding sites.
Enzyme binding sites may be derived from promoter proteins,
restriction enzymes, and the like. See, e.g., Stryer, L. (1988)
Biochemistry, W. H. Freeman, Palo Alto. Antibodies will typically
be produced using standard procedures, see, e.g., Harlow and Lane
(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press,
New York; and Goding (1986) Monoclonal Antibodies: Principles and
Practice, (2d Ed.) Academic Press, San Diego.
Typically, an antigen, or collection of antigens are presented to
an immune system. This may take the form of synthesized short
polymers produced by the VLSIPS technology, or by the other
synthetic means, or from isolation of natural products. For
example, antigen for the polypeptides may be made by the VLSIPS
technology, by standard peptide synthesis, by isolation of natural
proteins with or without degradation to shorter segments, or by
expression of a collection of short nucleic acids of random or
defined sequences. See, e.g., Tuerk and Gold (1990) Science
249:505-510, for generation of a collection of randomly mutagenized
oligonucleotides useful for expression.
The antigen or collection is presented to an appropriate immune
system, e.g., to a whole animal as in a standard immunization
protocol, or to a collection of immune cells or equivalent. In
particular, see Ward et al. (1989) Nature 341:544-546; and Huse et
al. (1989) Science 246:1275-1281, each of which is hereby
incorporated herein by reference.
A large diversity of antibodies will be generated, some of which
have specificities for the desired sequences. Antibodies may be
purified having the desired sequence specificities by isolating the
cells producing them. For example, a VLSIPS substrate with the
desired antigens synthesized thereon may be used to isolate cells
with cell surface reagents which recognize the antigens. The VLSIPS
substrate may be used as an affinity reagent to select and recover
the appropriate cells. Antibodies from those cells may be attached
to a substrate using the caged biotin methodology, or by attaching
a targeting molecule, e.g., an oligonucleotide. Alternatively, the
supernatants from antibody producing cells can be easily assayed
using a VLSIPS substrate to identify the cells producing the
appropriate antibodies.
Although cells may be isolated, specific antibody molecules which
perform the sequence recognition will also be sufficient.
Preferably populations of antibody with a known specificity can be
isolated. Supernatants from a large population of producing cells
may be passed over a VLSIPS substrate to bind to the desired
antigens attached to the substrate. When a sufficient density of
antibody molecules are attached, they may be removed by an
automated process, preferably as antibody populations exhibiting
specificity of binding.
In one particular embodiment, a VLSIPS substrate, e.g., with a
large plurality of fingerprint antigens attached thereto, is used
to isolate antibodies from a supernatant of a population of cells
producing antibodies to the antigens. Using the substrate as an
affinity reagent, the antibodies will attach to the appropriate
positionally defined antigens. The antibodies may be carefully
removed therefrom, preferably by an automated system which retains
their homogeneous specificities. The isolated antibodies can be
attached to a new substrate in a positionally defined matrix
pattern.
In a further embodiment, these spatially separated antibodies may
be isolated using a specific targeting method for isolation. In
this embodiment, a linker molecule which attaches to a particular
portion of the antibody, preferably away from the binding site, can
be attached to the antibodies. Various reagents will be used,
including staphylococcus protein A or antibodies which bind to
domains remote from the binding site. Alternatively, the antibodies
in the population, before affinity purification, may be derivatized
with an appropriate reagent compatible with new VLSIPS synthesis. A
preferred reagent is a nucleotide which can serve as a linker to
synthetic VLSIPS steps for synthesizing a specific sequence
thereon. Then, by successive VLSIPS cycles, each of the antibodies
attached to the defined antigen regions can have a defined
oligonucleotide synthesized thereon and corresponding in area to
the region of the substrate having each antigen attached. These
defined oligonucleotides will be useful as targeting reagents to
attach those antibodies possessing the same target sequence
specificity at defined positions on a new substrate, by virtue of
having bound to the antigen region, to a new VLSIPS substrate
having the complementary target oligonucleotides positionally
located on it. In this fashion, a VLSIPS substrate having the
desired antigens attached thereto can be used to generate a second
VLSIPS substrate with positionally defined reagents which recognize
those antigens.
The selected antigens will typically be selected to be those which
define particular functionalities or properties, so as to be useful
for fingerprinting and other uses. They will also be useful for
mapping and sequencing embodiments.
IV. FINGERPRINTING
A. General
Many of the procedures and techniques used in the polynucleotide
sequencing section are also appropriate for fingerprinting
applications. See, e.g., Poustka, et al. (1986) Cold Spring Harbor
Symposia on Quant. Biol., vol. LI, 131-139, Cold Spring Harbor
Press, New York; which is hereby incorporated herein by reference.
The fingerprinting method provided herein is based, in part, upon
the ability to positionally localize a large number of different
specific probes onto a single substrate. This high density matrix
pattern provides the ability to screen for, or detect, a very large
number of different sequences simultaneously. In fact, depending
upon the hybridization conditions, fingerprinting to the resolution
of virtually absolute matching of sequence is possible thereby
approaching an absolute sequencing embodiment. And the sequencing
embodiment is very useful in identifying the probes useful in
further fingerprinting uses. For example, characteristic features
of genetic sequences will be identified as being diagnostic of the
entire sequence. However, in most embodiments, longer probe and
target will be used, and for which slight mismatching may not need
to be resolved.
B. Preparation of Substrate Matrix
A collection of specific probes may be produced by either of the
methods described above in the section on sequencing. Specific
oligonucleotide probes of desired lengths may be individually
synthesized on a standard oligonucleotide synthesizer. The length
of these probes is limited only by the ability of the synthesizer
to continue to accurately synthesize a molecule. Oligonucleotides
or sequence fragments may also be isolated from natural sources.
Biological amplification methods may be coupled with synthetic
synthesizing procedures such as, e.g., polymerase chain
reaction.
In one embodiment, the individually isolated probes may be attached
to the matrix at defined positions. These probe reagents may be
attached by an automated process making use of the caged biotin
methodology described in Ser. No. 07/612,671, or using
photochemical reagents, see, e.g., Dattagupta et al. (1985) U.S.
Pat. No. 4,542,102 and (1987) U.S. Pat. No. 4,713,326. Each
individually purified reagent can be attached individually at
specific locations on a substrate.
In another embodiment, the VLSIPS synthesizing technique may be
used to synthesize the desired probes at specific positions on a
substrate. The probes may be synthesized by successively adding
appropriate monomer subunits, e.g., nucleotides, to generate the
desired sequences.
In another embodiment, a relatively short specific oligonucleotide
is used which serves as a targeting reagent for positionally
directing the sequence recognition reagent. For example, the
sequence specific reagents having a separate additional sequence
recognition segment (usually of a different polymer from the target
sequence) can be directed to target oligonucleotides attached to
the substrate. By use of non-natural targeting reagents, e.g.,
unusual nucleotide analogues which pair with other unnatural
nucleotide analogues and which do not interfere with natural
nucleotide interactions, the natural and non-natural portions can
coexist on the same molecule without interfering with their
individual functionalities. This can combine both a synthetic and
biological production system analogous to the technique for
targeting monoclonal antibodies to locations on a VLSIPS substrate
at defined positions. Unnatural optical isomers of nucleotides may
be useful unnatural reagents subject to similar chemistry, but
incapable of interfering with the natural biological polymers. See
also, Ser. No. 07/626,730, which is hereby incorporated herein by
reference.
After the separate substrate attached reagents are attached to the
targeting segment, the two are crosslinked, thereby permanently
attaching them to the substrate. Suitable crosslinking reagents are
known, see, e.g., Dattagupta et al. (1985) U.S. Pat. No. 4,542,102
and (1987) "Coupling of nucleic acids to solid support by
photochemical methods," U.S. Pat. No. 4,713,326, each of which is
hereby incorporated herein by reference. Similar linkages for
attachment of proteins to a solid substrate are provided, e.g., in
Merrifield (1986) Science 232:341-347, which is hereby incorporated
herein by reference.
C. Labeling Target Nucleotides
The labeling procedures used in the sequencing embodiments will
also be applicable in the fingerprinting embodiments. However,
since the fingerprinting embodiments often will involve relatively
large target molecules and relatively short oligonucleotide probes,
the amount of signal necessary to incorporate into the target
sequence may be less critical than in the sequencing applications.
For example, a relatively long target with a relatively small
number of labels per molecule may be easily amplified or detected
because of the relatively large target molecule size.
In various embodiments, it may be desired to cleave the target into
smaller segments as in the sequencing embodiments. The labeling
procedures and cleavage techniques described in the sequencing
embodiments would usually also be applicable here.
D. Hybridization Conditions
The hybridization conditions used in fingerprinting embodiments
will typically be less critical than for the sequencing
embodiments. The reason is that the amount of mismatching which may
be useful in providing the fingerprinting information would
typically be far greater than that necessary in sequencing uses.
For example, Southern hybridizations do not typically distinguish
between slightly mismatched sequences. Under these circumstances,
important and valuable information may be arrived at with less
stringent hybridization conditions while providing valuable
fingerprinting information. However, since the entire substrate is
typically exposed to the target molecule at one time, the binding
affinity of the probes should usually be of approximately
comparable levels. For this reason, if oligonucleotide probes are
being used, their lengths should be approximately comparable and
will be selected to hybridize under conditions which are common for
most of the probes on the substrate. Much as in a Southern
hybridization, the target and oligonucleotide probes are of lengths
typically greater than about 25 nucleotides. Under appropriate
hybridization conditions, e.g., typically higher salt and lower
temperature, the probes will hybridize irrespective of imperfect
complementarity. In fact, -with probes of greater than, e.g., about
fifty nucleotides, the difference in stability of different sized
probes will be relatively minor.
Typically the fingerprinting is merely for probing similarity or
homology. Thus, the stringency of hybridization can usually be
decreased to fairly low levels. See, e.g., Wetmur and Davidson
(1968) "Kinetics of Renaturation of DNA," J. Mol. Biol.,
31:349-370; and Kanehisa, M. (1984) Nuc. Acids Res.,
12:203-213.
E. Detection; VLSIPS.TM. Technology Scanning
Detection methods will be selected which are appropriate for the
selected label. The scanning device need not necessarily be
digitized or placed into a specific digital database, though such
would most likely be done. For example, the analysis in
fingerprinting could be photographic. Where a standardized
fingerprint substrate matrix is used, the pattern of hybridizations
may be spatially unique and may be compared photographically. In
this manner, each sample may have a characteristic pattern of
interactions and the likelihood of identical patterns will
preferably be such low frequency that the fingerprint pattern
indeed becomes a characteristic pattern virtually as unique as an
individual's fingertip fingerprint. With a standardized substrate,
every individual could be, in theory, uniquely identifiable on the
basis of the pattern of hybridizing to the substrate.
Of course, the VLSIPS.TM. Technology scanning apparatus may also be
useful to generate a digitized version of the fingerprint pattern.
In this way, the identification pattern can be provided in a linear
string of digits. This sequence could also be used for a
standardized identification system providing significant useful
medical transferability of specific data. In one embodiment, the
probes used are selected to be of sufficiently high resolution to
measure the antigens of the major histo compatibility complex. It
might even be possible to provide transplantation matching data in
a linear stream of data. The fingerprinting data may provide a
condensed version, or summary, of the linear genetic data, or any
other information data base.
F. Analysis
The analysis of the fingerprint will often be much simpler than a
total sequence determination. However, there may be particular
types of analysis which will be substantially simplified by a
selected group of probes. For example, probes which exhibit
particular populational heterogeneity may be selected. In this way,
analysis may be simplified and practical utility enhanced merely by
careful selection of the specific probes and a careful matrix
layout of those probes.
G. Substrate Reuse
As with the sequencing application, the fingerprinting usages may
also take advantage of the reusability of the substrate. In this
way, the interactions can be disrupted, the substrate treated, and
the renewed substrate is equivalent to an unused substrate.
H. Non-polynucleotide Aspects
Besides polynucleotide applications, the fingerprinting analysis
may be applied to other polymers, especially polypeptides,
carbohydrates, and other polymers, both organic and inorganic.
Besides using the fingerprinting method for analyzing a particular
polymer, the fingerprinting method may be used to characterize
various samples. For example, a cell or population of cells may be
tested for their expression of specific antigens or their mRNA
sequence intent. For example, a T-cell may be classified by virtue
of its combination of expressed surface antigens. With specific
reagents which interact with these antigens, a cell or a population
of cells or a lysed cell may be exposed to a VLSIPS substrate. The
biological sample may be classified or characterized by analyzing
the pattern of specific interaction. This may be applicable to a
cell or tissue type, to the messenger RNA population expressed by a
cell to the genetic content of a cell, or to virtually any sample
which can be classified and/or identified by its combination of
specific molecular properties.
The ability to generate a high density means for screening the
presence or absence of specific interactions allows for the
possibility of screening for, if not saturating, all of a very
large number of possible interactions. This is very powerful in
providing the means for testing the combinations of molecular
properties which can define a class of samples. For example, a
species of organism may be characterized by its DNA sequences,
e.g., a genetic fingerprint. By using a fingerprinting method, it
may be determined that all members of that species are sufficiently
similar in specific sequences that they can be easily identified as
being within a particular group. Thus, newly defined classes may be
resolved by their similarity in fingerprint patterns.
Alternatively, a non-member of that group will fail to share those
many identifying characteristics. However, since the technology
allows testing of a very large number of specific interactions, it
also provides the ability to more finely distinguish between
closely related different cells or samples. This will have
important applications in diagnosing viral, bacterial, and other
pathological on nonpathological infections.
In particular, cell classification may be defined by any of a
number of different properties. For example, a cell class may be
defined by its DNA sequences contained therein. This allows species
identification for parasitic or other infections. For example, the
human cell is presumably genetically distinguishable from a monkey
cell, but different human cells will share many genetic markers. At
higher resolution, each individual human genome will exhibit unique
sequences that can define it as a single individual.
Likewise, a developmental stage of a cell type may be definable by
its pattern of expression of messenger RNA. For example, in
particular stages of cells, high levels of ribosomal RNA are found
whereas relatively low levels of other types of messenger RNAs may
be found. The high resolution distinguishability provided by this
fingerprinting method allows the distinction between cells which
have relatively minor differences in its expressed mRNA population.
Where a pattern is shown to be characteristic of a stage, a stage
may be defined by that particular pattern of messenger RNA
expression.
In a similar manner, the antigenic determinants found on a protein
may very well define the cell class. For example, immunological
T-cells are distinguishable from B-cells because, in part, the cell
surface antigens on the cell types are distinguishable. Different
T-cell subclasses can be also distinguished from one another by
whether they contain particular T-cell antigens. The present
invention provides the possibility for high resolution testing of
many different interactions simultaneously, and the definition of
new cell types will be possible.
The high resolution VLSIPS.TM. substrate may also be used as a very
powerful diagnostic tool to test the combination of presence, of a
plurality of different assays from a biological sample. For
example, a cancerous condition may be indicated by a combination of
various different properties found in the blood. For example, a
cancerous condition may be indicated by a combination of expression
of various soluble antigens found in the blood along with a high
number of various cellular antigens found on lymphocytes and/or
particular cell degradation products. With a substrate as provided
herein, a large number of different features can be simultaneously
performed on a biological sample. In fact, the high resolution of
the test will allow more complete characterization of parameters
which define particular diseases. Thus, the power of diagnostic
tests may be limited by the extent of statistical correlation with
a particular condition rather than with the number of antigens or
interactions which are tested. The present invention provides the
means to generate this large universe of possible reagents and the
ability to actually accumulate that correlative data.
In another embodiment, a substrate as provided herein may be used
for genetic screening. This would allow for simultaneous screening
of thousands of genetic markers. As the density of the matrix is
increased, many more molecules can be simultaneously tested.
Genetic screening then becomes a simpler method as the present
invention provides the ability to screen for thousands, tens
of-thousands, and hundreds of thousands, even millions of different
possible genetic features. However, the number of high correlation
genetic markers for conditions numbers only in the hundreds. Again,
the possibility for screening a large number of sequences provides
the opportunity for generating the data which can provide
correlation between sequences and specific conditions or
susceptibility. The present invention provides the means to
generate extremely valuable correlations useful for the genetic
detection of the causative mutation leading to medical conditions.
In still another embodiment, the present invention would be
applicable to distinguishing two individuals having identical
genetic compositions. The antibody population within an individual
is dependent both on genetic and historical factors. Each
individual experiences a unique exposure to various infectious
agents, and the combined antibody expression is partly determined
thereby. Thus, individuals may also be fingerprinted by their
immunological content, either of actively expressed antibodies, or
their immunological memory. Similar sorts of immunological and
environmental histories may be useful for fingerprinting, perhaps
in combination with other screening properties. In particular, the
present invention may be useful for screening allergic reactions or
susceptibilities, and a simple IgE specificity test may be useful
in determining a spectrum of allergies.
With the definition of new classes of cells, a cell sorter will be
used to purify them. Moreover, new markers for defining that class
of cells will be identified. For example, where the class is
defined by its RNA content, cells may be screened by antisense
probes which detect the presence or absence of specific sequences
therein. Alternatively, cell lysates may provide information useful
in correlating intracellular properties with extracellular markers
which indicate functional differences, Using standard cell sorter
technology with a fluorescence or labeled antisense probe which
recognizes the internal presence of the specific sequences of
interest, the cell sorter will be able to isolate a relatively
homogeneous population of cells possessing the particular marker.
Using successive probes the sorting process should be able to
select for cells having a combination of a large number of
different markers.
In a non-polynucleotide embodiment, cells may be defined by the
presence of other markers. The markers may be carbohydrates,
proteins, or other molecules. Thus, a substrate having particular
specific reagents, e.g., antibodies, attached to it should be able
to identify cells having particular patterns of marker expression.
Of course, combinations of these made be utilized and a cell class
may be defined by a combination of its expressed mRNA, its
carbohydrate expression, its antigens, and other properties. This
fingerprinting should be useful in determining the physiological
state of a cell or population of cells.
Having defined a cell type whose function or properties are defined
by the reagents attachable to a VLSIPS substrate, such as cellular
antigens, these structural manifestations of function may be used
to sort cells to generate a relatively homogeneous population of
that class of cells. Standard cell sorter technology may be applied
to purify such a population, see, e.g., Dangl, J. and Herzenberg
(1982) "Selection of hybridomas and hybridoma variants using the
fluorescence activated cell sorter," J. Immunological Methods
52:1-14; and Becton Dickinson, Fluorescence Activated Cell Sorter
Division, San Jose, Calif., and Coulter Diagnostics, Hialeah,
Fla.
With the fingerprinting method an identification means arises from
mosaicism problems in an organism. A mosaic organism is one whose
genetic content in different cells is significantly different.
Various clonal populations should have similar genetic
fingerprints, though different clonal populations may have
different genetic contents. See, for example, Suzuki et al. An
Introduction to Genetic Analysis (4th Ed.), Freeman and Co., New
York, which is hereby incorporated herein by reference. However,
this problem should be a relatively rare problem and could be more
carefully evaluated with greater experience using the
fingerprinting methods.
The invention will also find use in detecting changes, both genetic
and antigenic, e.g., in a rapidly "evolving" protozoa infection, or
similarly changing organism.
V. MAPPING
A. General
The use of the present invention for mapping parallels its use for
fingerprinting and sequencing. Where a polymer is a linear
molecule, the mapping provides the ability to locate particular
segments along the length of the polymer. Branched polymers can be
treated as a series of individual linear polymers. The mapping
provides the ability to locate, in a relative sense, the order of
various subsequences. This may be achieved using at least two
different approaches.
The first approach is to take the large sequence and fragment it at
specific points. The fragments are then ordered and attached to a
solid substrate. For example, the clones resulting from a
chromosome walking process may be individually attached to the
substrate by methods, e.g., caged biotin techniques, indicated
earlier. Segments of unknown map position will be exposed to the
substrate and will hybridize to the segment which contains that
particular sequence. This procedure allows the rapid determination
of a number of different labeled segments, each mapping requiring
only a single hybridization step once the substrate is generated.
The substrate may be regenerated by removal of the interaction, and
the next mapping segment applied.
In an alternative method, a plurality of subsequences can be
attached to a substrate. Various short probes may be applied to
determine which segments may contain particular overlaps. The
theoretical basis and a description of this mapping procedure is
contained in, e.g., Evans et al. 1989 "Physical Mapping of Complex
Genomes by Cosmid Multiplex Analysis," Proc. Natl. Acad. Sci. USA
86:5030-5034, and other references cited above in the Section
labeled "Overall Description." Using this approach, the details of
the mapping embodiment are very similar to those used in the
fingerprinting embodiment.
B. Preparation of Substrate Matrix
The substrate may be generated in either of the methods generally
applicable in the sequencing and fingerprinting embodiments. The
substrate may be made either synthetically, or by attaching
otherwise purified probes or sequences to the matrix. The probes or
sequences may be derived either from synthetic or biological means.
As indicated above, the solid phase substrate synthetic methods may
be utilized to generate a matrix with positionally defined
sequences. In the mapping embodiment, the importance of saturation
of all possible subsequences of a preselected length is far less
important than in the sequencing embodiment, but the length of the
probes used may be desired to be much longer. The processes for
making a substrate which has longer oligonucleotide probes should
not be significantly different from those described for the
sequencing embodiments, but the optimization parameters may be
modified to comply with the mapping needs.
C. Labeling
The labeling methods will be similar to those applicable in
sequencing and fingerprinting embodiments. Again, it may be
desirable to fragment the target sequences.
D. Hybridization/Specific Interaction
The specificity of interaction between the targets and probe would
typically be closer to those used for fingerprinting embodiments,
where homology is more important than absolute distinguishability
of high fidelity complementary hybridization. Usually, the
hybridization conditions will be such that merely homologous
segments will interact and provide a positive signal. Much like the
fingerprinting embodiment, it may be useful to measure the extent
of homology by successive incubations at higher stringency
conditions. Or, a plurality of different probes, each having
various levels of homology may be used. In either way, the spectrum
of homologies can be measured.
Where non-nucleic acid hybridization is involved, the specific
interactions may also be compared in a fingerprint-like manner. The
specific reagents may have less specificity, e.g., monoclonal
antibodies which recognize a broader spectrum of sequences may be
utilized relative to a sequencing embodiment. Again, the
specificity of interaction may be measured under various conditions
of increasing stringency to determine the spectrum of matching
across the specific probes selected, or a number of different
stringency reagents may be included to indicate the binding
affinity.
E. Detection
The detection methods used in the mapping procedure will be
virtually identical to those used in the fingerprinting embodiment.
The detection methods will be selected in combination with the
labeling methods.
F. Analysis
The analysis of the data in a mapping embodiment will typically be
somewhat different from that in fingerprinting. The fingerprinting
embodiment will test for the presence or absence of specific or
homologous segments. However, in the mapping embodiment, the
existence of an interaction is coupled with some indication of the
location of the interaction. The interaction is mapped in some
manner to the physical polymer sequence. Some means for determining
the relative positions of different probes is performed. This may
be achieved by synthesis of the substrate in pattern, or may result
from analysis of sequences after they have been attached to the
substrate.
For example, the probes may be randomly positioned at various
locations on the substrate. However, the relative positions of the
various reagents in the original polymer may be determined by using
short fragments, e.g., individually, as target molecules which
determine the proximity of different probes. By an automated system
of testing each different short fragment of the original polymer,
coupled with proper analysis, it will be possible to determine
which probes are adjacent one another on the original target
sequence and correlate that with positions on the matrix. In this
way, the matrix is useful for determining the relative locations of
various new segments in the original target molecule. This sort of
analysis is described in Evans, and the related references
described above.
G. Substrate Reuse
The substrate should be reusable in the manner described in the
fingerprinting section. The substrate is renewed by removal of the
specific interactions and is washed and prepared for successive
cycles of exposure to new target sequences.
H. Non-polynucleotide Aspects
The mapping procedure may be used on other molecules than
polynucleotides. Although hybridization is one type of specific
interaction which is clearly useful for use in this mapping
embodiment, antibody reagents may also be very useful. In the same
way that polypeptide sequencing or other polymers may be sequenced
by the reagents and techniques described in the sequencing section
and fingerprinting section, the mapping embodiment may also be used
similarly.
In another form of mapping, as described above in the
fingerprinting section, the developmental map of a cell or
biological system may be measured using fingerprinting type
technology. Thus, the mapping may be along a temporal dimension
rather than along a polymer dimension. The mapping or
fingerprinting embodiments may also be used in determining the
genetic rearrangements which may be genetically important, as in
lymphocyte and B-cell development. In another example, various
rearrangements or chromosomal dislocations may be tested by either
the fingerprinting or mapping methods. These techniques are similar
in many respects and the fingerprinting and mapping embodiments may
overlap in many respects.
VI. ADDITIONAL SCREENING AND APPLICATIONS
A. Specific Interactions
As originally indicated in the parent filing of VLSIPS.TM.
Technology, the production of a high density plurality of spatially
segregated polymers provides the ability to generate a very large
universe or repertoire of individually and distinct sequence
possibilities. As indicated above, particular oligonucleotides may
be synthesized in automated fashion at specific locations on a
matrix. In fact, these oligonucleotides may be used to direct other
molecules to specific locations by linking specific
oligonucleotides to other reagents which are in batch exposed to
the matrix and hybridized in a complementary fashion to only those
locations where the complementary oligonucleotide has been
synthesized on the matrix. This allows for spatially attaching a
plurality of different reagents onto the matrix instead of
individually attaching each separate reagent at each specific
location. Although the caged biotin method allows automated
attachment, the speed of the caged biotin attachment process is
relatively slow and requires a separate reaction for each reagent
being attached. By use of the oligonucleotide method, the
specificity of position can be done in an automated and parallel
fashion. As each reagent is produced, instead of directly attaching
each reagent at each desired position, the reagent may be attached
to a specific desired complementary oligonucleotide which will
ultimately be specifically directed toward locations on the matrix
having a complementary oligonucleotide attached thereat.
In addition, the technology allows screening for specificity of
interaction with particular reagents. For example, the
oligonucleotide sequence specificity of binding of a potential
reagent may be tested by presenting to the reagent all of the
possible subsequences available for binding. Although secondary or
higher order sequence specific features might not be easily
screenable using this technology, it does provide a convenient,
simple, quick, and thorough screen of interactions between a
reagent and its target recognition sequences. See, e.g., Pfeifer et
al. (1989) Science 246:810-812.
For example, the interaction of a promoter-protein with its target
binding sequence may be tested for many different, or all, possible
binding sequences. By testing the strength of interactions under
various different conditions, the interaction of the promoter
protein with each of the different potential binding sites may be
analyzed. The spectrum of strength of interactions with each
different potential binding site may provide significant insight
into the types of features which are important in determining
specificity.
An additional example of a sequence specific interaction between
reagents is the testing of binding of a double stranded nucleic
acid structure with a single stranded oligonucleotide. Often, a
triple stranded structure is produced which has significant aspects
of sequence specificity.
Testing of such interactions with either sequences comprising only
natural nucleotides, or perhaps the testing of nucleotide analogs
may be very important in screening for particularly useful
diagnostic or therapeutic reagents. See, e.g., Haner and Dervan
(1990) Biochemistry 29:9761-6765, and references therein.
B. Sequence Comparisons
Once a gene is sequenced, the present invention provides a means to
compare alleles or related sequences to locate and identify
differences from the control sequence. This would be extremely
useful in further analysis of genetic variability at a specific
gene locus.
C. Categorizations
As indicated above in the fingerprinting and mapping embodiments,
the present invention is also useful in defining specific stages in
the temporal sequence of cells, e.g., development, and the
resulting tissues within an organism. For example, the
developmental stage of a cell, or population of cells, can be
dependent upon the expression of particular messenger RNAs or
cellular antigens. The screening procedures provided allow for high
resolution definition of new classes of cells. In addition, the
temporal development of particular cells will be characterized by
the presence or expression of various mRNAs. Means to
simultaneously screen a plurality or very large number of different
sequences are provided. The combination of different markers made
available dramatically increases the ability to distinguish fairly
closely related cell types. Other markers may be combined with
markers and methods made available herein to define new
classifications of biological samples, e.g., based upon new
combinations of markers.
The presence or absence of particular marker sequences will be used
to define temporal developmental stages. Once the stages are
defined, fairly simple methods can be applied to actually purify
those particular cells. For example, antisense probes or
recognition reagents may be used with a cell sorter to select those
cells containing or expressing the critical markers. Alternatively,
the expression of those sequences may result in specific antigens
which may also be used in defining cell classes and sorting those
cells away from others. In this way, for example, it should be
possible to select a class of omnipotent immune system cells which
are able to completely regenerate a human immune system. Based upon
the cellular classes defined by the parameters made available by
this technology, purified classes of cells having identifiable
differences, structural or functional, are made available.
In an alternative embodiment, a plurality of antigens or specific
binding proteins attached to the substrate may be used to define
particular cell types. For example, subclasses of T-cells are
defined, in part, by the combination of expressed cell surface
antigens. The-present invention allows for the simultaneous
screening of a large plurality of different antigens together.
Thus, higher resolution classification of different T-cell
subclasses becomes possible and, with the definitions and
functional differences which correlate with those antigenic or
other parameters, the ability to purify those cell types becomes
available. This is applicable not only to T-cells, but also to
lymphocyte cells, or even to freely circulating cells. Many of the
cells for which this would be most useful will be immobile cells
found in particular tissues or organs. Tumor cells will be
diagnosed or detected using these fingerprinting techniques.
Coupled with a temporal change in structure, developmental classes
may also be selected and defined using these technologies. The
present invention also provides the ability not only to define new
classes of cells based upon functional or structural differences,
but it also provides the ability to select or purify populations of
cells which share these particular properties. Standard cell
sorting procedures using antibody markers may be used to detect
extracellular features. Intracellular features would also be
detectable by introducing the label reagents into the cell. In
particular, antisense DNA or RNA molecules may be introduced into a
cell to detect RNA sequences therein. See, e.g., Weintraub (1990)
Scientific American 262:40-46.
D. Statistical Correlations
In an additional embodiment, the present invention also allows for
the high resolution correlation of medical conditions with various
different markers. For example, the presently available technology,
when applied to amniocentesis or other genetic screening methods,
typically screens for tens of different markers at most. The
present invention allows simultaneous screening for tens, hundreds,
thousands, tens of thousands, hundreds of thousands, and even
millions of different genetic sequences. Thus, applying the
fingerprinting methods of the present invention to a sufficiently
large population allows detailed statistical analysis to be made,
thereby correlating particular medical conditions with particular
markers, typically antigenic or genetic. Tumor specific antigens
will be identified using the present invention.
Various medical conditions may be correlated against an enormous
data base of the sequences within an individual. Genetic
propensities and correlations then become available and high
resolution genetic predictability and correlation become much more
easily performed. With the enormous data base, the reliability of
the predictions is also better tested. Particular markers which are
partially diagnostic of particular medical conditions or medical
susceptibilities will be identified and provide direction in
further studies and more careful analysis of the markers involved.
Of course, as indicated above in the sequencing embodiment, the
present invention will find much use in intense sequencing
projects. For example, sequencing of the entire human genome in the
human genome project will be greatly simplified and enabled by the
present invention.
VI. FORMATION OF SUBSTRATE
The substrate is provided with a pattern of specific reagents which
are positionally localized on the surface of the substrate. This
matrix of positions is defined by the automated system which
produces the substrate. The instrument will typically be one
similar to that described in Pirrung et al. (1992) U.S. Pat. No.
5,143,854, and Ser. No. 07/624,120, now abandoned. The
instrumentation described therein is directly applicable to the
applications used here. In particular, the apparatus comprises a
substrate, typically a silicon containing substrate, on which
positions on the surface may be defined by a coordinate system of
positions. These positions can be individually addressed or
detected by the VLSIPS.TM. Technology apparatus.
Typically, the VLSIPS.TM. Technology apparatus uses optical methods
used in semiconductor fabrication applications. In this way, masks
may be used to photo-activate positions for attachment or synthesis
of specific sequences on the substrate. These manipulations may be
automated by the types of apparatus described in Pirrung et al.
(1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120, now
abandoned.
Selectively removable protecting groups allow creation of well
defined areas of substrate surface having differing reactivities.
Preferably, the protecting groups are selectively removed from the
surface by applying a specific activator, such as electromagnetic
radiation of a specific wavelength and intensity. More preferably,
the specific activator exposes selected areas of surface to remove
the protecting groups in the exposed areas.
Protecting groups of the present invention are used in conjunction
with solid phase oligomer syntheses, such as peptide syntheses
using natural or unnatural amino acids, nucleotide syntheses using
deoxyribonucleic and ribonucleic acids, oligosaccharide syntheses,
and the like. In addition to protecting the substrate surface from
unwanted reaction, the protecting groups block a reactive end of
the monomer to prevent self-polymerization. For instance,
attachment of a protecting group to the amino terminus of an
activated amino acid, such as the N-hydroxysuccinimide-activated
ester of the amino acid prevents the amino terminus of one monomer
from reacting with the activated ester portion of another during
peptide synthesis.
Alternatively, the protecting group may be attached to the carboxyl
group of an amino acid to prevent reaction at this site. Most
protecting groups can be attached to either the amino or the
carboxyl group of an amino acid, and the nature of the chemical
synthesis will dictate which reactive group will require a
protecting group. Analogously, attachment of a protecting group to
the 5'-hydroxyl group of a nucleoside during synthesis using for
example, phosphate-triester coupling chemistry, prevents the
5'-hydroxyl of one nucleoside from reacting with the 3'-activated
phosphate-triester of another.
Regardless of the specific use, protecting groups are employed to
protect a moiety on a molecule from reacting with another reagent.
Protecting groups of the present invention have the following
characteristics: they prevent selected reagents from modifying the
group to which they are attached; they are stable (that is, they
remain attached) to the synthesis reaction conditions; they are
removable under conditions that do not adversely affect the
remaining structure; and once removed, do not react appreciably
with the surface or surface-bound oligomer. The selection of a
suitable protecting group will depend, of course, on the chemical
nature of the monomer unit and oligomer, as well as the specific
reagents they are to protect against.
In a preferred embodiment, the protecting groups will be
photoactivatable. The properties and uses of photoreactive
protecting compounds have been reviewed. See, McCray et al., Ann.
Rev. of Biophys. and Biophys. Chem. (1989) 18:239-270, which is
incorporated herein by reference. Preferably, the photosensitive
protecting groups will be removable by radiation in the ultraviolet
(UV) or visible portion of the electromagnetic spectrum. More
preferably, the protecting groups will be removable by radiation in
the near UV or visible portion of the spectrum. In some
embodiments, however, activation may be performed by other methods
such as localized heating, electron beam lithography, laser
pumping, oxidation or reduction with microelectrodes, and the like.
Sulfonyl compounds are suitable reactive groups for electron beam
lithography. Oxidative or reductive removal is accomplished by
exposure of the protecting group to an electric current source,
preferably using microelectrodes directed to the predefined regions
of the surface which are desired for activation. A more detailed
description of these protective groups is provided in Ser. No.
07/624,120, now abandoned, which is hereby incorporated herein by
reference.
The density of reagents attached to a silicon substrate may be
varied by standard procedures. The surface area for attachment of
reagents may be increased by modifying the silicon surface. For
example, a matte surface may be machined or etched on the substrate
to provide more sites for attachment of the particular reagents.
Another way to increase the density of reagent binding sites is to
increase the derivitization density of the silicon. Standard
procedures for achieving this are described, below.
One method to control the derivatization density is to highly
derivatize the substrate with photochemical groups at high density.
The substrate is then photolyzed for various predetermined times,
which photoactivate the groups at a measurable rate, and react them
with a capping reagent. By this method, the density of linker
groups may be modulated by using a desired time and intensity of
photoactivation.
In many applications, the number of different sequences which may
be provided may be limited by the density and the size of the
substrate on which the matrix pattern is generated. In situations
where the density is insufficiently high to allow the screening of
the desired number of sequences, multiple substrates may be used to
increase the number of sequences tested. Thus, the number of
sequences tested may be increased by using a plurality of different
substrates. Because the VLSIPS apparatus is almost fully automated,
increasing the number of substrates does not lead to a significant
increase in the number of manipulations which must be performed by
humans. This again leads to greater reproducibility and speed in
the handling of these multiple substrates.
A. Instrumentation
The concept of using VLSIPS.TM. Technology generally allows a
pattern or a matrix of reagents to be generated. The procedure for
making the pattern is performed by any of a number of different
methods. An apparatus and instrumentation useful for generating a
high density VLSIPS substrate is described in detail in Pirrung et
al. (1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120, now
abandoned.
B. Binary Masking
The details of the binary masking are described in an accompanying
application filed simultaneously with this, Ser. No. 07/624,120,
now abandoned, whose specification is incorporated herein by
reference.
For example, the binary masking technique allows for producing a
plurality of sequences based on the selection of either of two
possibilities at any particular location. By a series of binary
masking steps, the binary decision may be the determination, on a
particular synthetic cycle, whether or not to add any particular
one of the possible subunits. By treating various regions of the
matrix pattern in parallel, the binary masking strategy provides
the ability to carry out spatially addressable parallel
synthesis.
C. Synthetic Methods
The synthetic methods in making a substrate are described in the
parent application, Pirrung et al. (1992) U.S. Pat. No. 5,143,854.
The construction of the matrix pattern on the substrate will
typically be generated by the use of photosensitive reagents. By
use of photo-lithographic optical methods, particular segments of
the substrate can be irradiated with light to activate or
deactivate blocking agents, e.g., to protect or deprotect
particular chemical groups. By an appropriate sequence of
photo-exposure steps at appropriate times with appropriate masks
and with appropriate reagents, the substrates can have known
polymers synthesized at positionally defined regions on the
substrate. Methods for synthesizing various substrates are
described in Pirrung et al. (1992) U.S. Pat. No. 5,143,854 and Ser.
No. 07/624,120, now abandoned. By a sequential series of these
photo-exposure and reaction manipulations, a defined matrix pattern
of known sequences may be generated, and is typically referred to
as a VLSIPS.TM. Technology substrate. In the nucleic acid synthesis
embodiment, nucleosides used in the synthesis of DNA by photolytic
methods will typically be one of the two forms shown below:
##STR4##
In I, the photolabile group at the 5' position is abbreviated NV
(nitroveratryl) and in II, the group is abbreviated NVOC
(nitroveratryl oxycarbonyl). Although not shown in FIG. C, the
bases (adenine, cytosine, and guanine) contain exocyclic NH.sub.2
groups which must be protected during DNA synthesis. Thymine
contains no exocyclic NH.sub.2 and therefore requires no
protection. The standard protecting groups for these amines are
shown below: ##STR5##
Other amides of the general formula ##STR6## where R may be alkyl
or aryl have been used.
Another type of protecting group FMOC (9-fluorenyl methoxycarbonyl)
is currently being used to protect the exocyclic amines of the
three bases: ##STR7##
The advantage of the FMOC group is that it is removed under mild
conditions (dilute organic bases) and can be used for all three
bases. The amide protecting groups require more
Nucleosides used as 5'-OH probes, useful in verifying correct
VLSIPS synthetic function, include, for example, the following:
##STR8##
These compounds are used to detect where on a substrate photolysis
has occurred by the attachment of either III or V to the newly
generated 5'-OH. In the case of III, after the phosphate attachment
is made, the substrate is treated with a dilute base to remove the
FMOC group. The resulting amine can be reacted with FITC and the
substrate examined by fluorescence microscopy. This indicates the
proper generation of a 5'-OH. In the case of compound IV, after the
phosphate attachment is made, the substrate is treated with FITC
labeled streptavidin and the substrate again may be examined by
fluorescence microscopy. Other probes, although not nucleoside
based, have included the following: ##STR9##
The method of attachment of the first nucleoside to the surface of
the substrate depends on the functionality of the groups at the
substrate surface. If the surface is amine functionalized, an amide
bond is made (see example below) ##STR10##
If the surface is hydroxy functionalized, a phosphate bond is made
(see example below) ##STR11##
In both cases, the thymidine example is illustrated, but any one of
the four phosphoramidite activated nucleosides can be used in The
first step.
Photolysis of the photolabile group NV or NVOC on the 5' positions
of the nucleosides is carried out at .sup..about. 362 nm with an
intensity of 14 mW/cm.sup.2 for 10 minutes with the substrate side
(side containing the photolabile group) immersed in dioxane. After
the coupling of the next nucleoside is complete, the photolysis is
repeated followed by another coupling until the desired oligomer is
obtained.
One of the most common 3'-O-protecting groups is the ester, in
particular the acetate: ##STR12##
The groups can be removed by mild base treatment 0.1N NaOH/MeOH or
K.sub.2 CO.sub.3 /H.sub.2 O/MeOH.
Another group used most often is the silyl ether: ##STR13##
These groups can be removed by neutral conditions using 1M
tetra-n-butylammonium fluoride in THF or under acid conditions.
With respect to photodeprotection, the nitroveratryl group could
also be used to protect the 3'-position. ##STR14##
Here, light (photolysis) would be used to remove these protecting
groups.
A variety of ethers can also be used in the protection of the
3'-O-position: ##STR15##
Removal of these groups usually involves acid or catalytic
methods.
Note that corresponding linkages and photoblocked amino acids are
described in detail in Ser. No. 07/624,120, now abandoned, which is
hereby incorporated herein by reference.
Although the specificity of interactions at particular locations
will usually be homogeneous due to a homogeneous polymer being
synthesized at each defined location, for certain purposes, it may
be useful to have mixed polymers with a commensurate mixed
collection of interactions occurring at specific defined locations,
or degeneracy reducing analogues, which have been discussed above
and show broad specificity in binding. Then, a positive interaction
signal may result from any of a number of sequences contained
therein.
As an alternative method of generating a matrix pattern on a
substrate, preformed polymers may be individually attached at
particular sites on the substrate. This may be performed by
individually attaching reagents one at a time to specific positions
on the matrix, a process which may be automated. See, e.g., Ser.
No. 07/435,316, now abandoned, and Barrett et al. (1993) U.S. Pat.
No. 5,252,743. Another way of generating a positionally defined
matrix pattern on a substrate is to have individually specific
reagents which interact with each specific position on the
substrate. For example, oligonucleotides may be synthesized at
defined locations on the substrate. Then the substrate would have
on its surface a plurality of regions having homogeneous
oligonucleotides attached at each position.
In particular, at least four different substrate preparation
procedures are available for treating a substrate surface. They are
the standard VLSIPS.TM. Technology method, polymeric substrates,
Durapore.TM., and synthetic beads or fibers. The treatment labeled
"standard VLSIPS.TM. Technology" method is described in Ser. No.
07/624,120, now abandoned, and involves applying
amino-propyltriethoxysilane to a glass surface.
The polymeric substrate approach involves either of two ways of
generating a polymeric substrate. The first uses a high
concentration of aminopropyltriethoxysilane (2-20%) in an aqueous
ethanol solution (95%). This allows the silane compound to
polymerize both in solution and on the substrate surface, which
provides a high density of amines on the surface of the glass. This
density is contrasted with the standard VLSIPS method. This
polymeric method allows for the deposition on the substrate surface
of a monolayer due to the anhydrous method used with the
aforementioned silane.
The second polymeric method involves either the coating or covalent
binding of an appropriate acrylic acid polymer onto the substrate
surface. In particular, e.g., in DNA synthesis, a monomer such as a
hydroxypropylacrylate is used to generate a high density of
hydroxyl groups on the substrate surface, allowing for the
formation of phosphate bonds. An example of such a compound is
shown: ##STR16##
The method using a Durapore.TM. membrane (Millipore) consists of a
polyvinylidine difluoride coating with crosslinked
polyhydroxylpropyl acrylate [PVDF-HPA]: ##STR17## Here the building
up of, e.g., a DNA oligomer, can be started immediately since
phosphate bonds to the surface can be accomplished in the first
step with no need for modification. A nucleotide dimer (5'-C-T-3')
has been successfully made on this substrate.
The fourth method utilizes synthetic beads or fibers. This would
use another substrate, such as a teflon copolymer graft bead or
fiber, which is covalently coated with an organic layer
(hydrophilic) terminating in hydroxyl sites (commercially available
from Molecular Biosystems, Inc.) This would offer the same
advantage as the Durapore.TM. membrane, allowing for immediate
phosphate linkages, but would give additional contour by the
3-dimensional growth of oligomers.
A matrix pattern of new reagents may be targeted to each specific
oligonucleotide position by attaching a complementary
oligonucleotide to which the substrate bound form is complementary.
For instance, a number of regions may have homogeneous
oligonucleotides synthesized at various locations. Oligonucleotide
sequences complementary to each of these can be individually
generated and linked to a particular specific reagents. Often these
specific reagents will be antibodies. As each of these is specific
for finding its complementary oligonucleotide, each of the specific
reagents will bind through the oligonucleotide to the appropriate
matrix position. A single step having a combination of different
specific reagents being attached specifically to a particular
oligonucleotide will thereby bind to its complement at the defined
matrix position. The oligonucleotides will typically then be
covalently attached, using, e.g., an acridine dye, for
photocrosslinking. Psoralen is a commonly used acridine dye for
photocrosslinking purposes, see, e.g., Song et al. (1979)
Photochem. Photobiol. 29:1177-1197; Cimino et al. (1985) Ann. Rev.
Biochem. 54:1151-1193; Parsons (1980) Photochem. Photobiol.
32:813-821; and Dattagupta et al. (1985) U.S. Pat. No. 4,542,102,
and (1987) U.S. Pat. No. 4,713,326; each of which is hereby
incorporated herein by reference. This method allows a single
attachment manipulation to attach all of the specific reagents to
the matrix at defined positions and results in the specific
reagents being homogeneously located at defined positions. In many
embodiments, the specific reagents will be antibodies.
In an alternative embodiment, antibody molecules may be used to
specifically direct binding to defined positions on a substrate.
The VLSIPS technology may be used to generate specific epitopes at
each position on the substrate. Antibody molecules having
specificity of interaction may be used to attach oligonucleotides,
thereby avoiding the interference of internal polynucleotide
sequences from binding to the substrate complementary
oligonucleotides. In fact, the specificity of interaction for
positional targeting may be achieved by use of nucleotide analogues
which do not interact with the natural nucleotides. For example,
other synthetic nucleotides have been made which undergo base
pairing, thereby providing the specificity of targeting, but the
synthetic nucleotides also do not interact with the natural
biological nucleotides. Thus, synthetic oligonucleotides would be
useful for attachment to biological nucleotides and specific
targeting. Moreover, the VLSIPS synthetic processes would be useful
in generating the VLSIPS substrate, and standard oligonucleotide
synthesis could be applied, with minor modifications, to produce
the complementary sequences which would be attached to other
specific reagents.
D. Surface Immobilization
1. Caged Biotin
An alternative method of attaching reagents in a positionally
defined matrix pattern is to use a caged biotin system. See Barrett
et al. (1993) U.S. Pat. No. 5,252,743, which is hereby incorporated
herein by reference, for additional details on the chemistry and
application of caged biotin embodiments. In short, the caged biotin
has a photosensitive blocking moiety which prevents the combination
of avidin to biotin. At positions where the photo-lithographic
process has removed the blocking group, high affinity biotin sites
are generated. Thus, by a sequential series of photolithographic
deblocking steps interspersed with exposure of those regions to
appropriate biotin containing reagents, only those locations where
the deblocking takes place will form an avidin-biotin interaction.
Because the avidin-biotin binding is very tight, this will usually
be virtually irreversible binding.
2. Crosslinked Interactions
The surface immobilization may also take place by photo
crosslinking of defined oligonucleotides linked to specific
reagents. After hybridization of the complementary
oligonucleotides, the oligonucleotides may be crosslinked by a
reagent by psoralen or another similar type of acridine dye. Other
useful cross linking reagents are described in Dattagupta et al.
(1985) U.S. Pat. No. 4,542,102, and (1987) U.S. Pat. No.
4,713,326.
In another embodiment, colony or phage plaque transfer of
biological polymers may be transferred directly onto a silicon
substrate. For example, a colony plate may be transferred onto a
substrate having a generic oligonucleotide sequence which
hybridizes to another generic complementary sequence contained on
all of the vectors into which inserts are cloned. This will
specifically only bind those molecules which are actually contained
in the vectors containing the desired complementary sequence. This
immobilization allows for producing a matrix onto which a sequence
specific reagent can bind, or for other purposes. In a further
embodiment, a plurality of different vectors each having a specific
oligonucleotide attached to the vector may be specifically attached
to particular regions on a matrix having a complementary
oligonucleotide attached thereto.
VIII. HYBRIDIZATION/SPECIFIC INTERACTION
A. General
As discussed previously in the VLSIPS.TM. Technology parent
applications, the VLSIPS.TM. technology substrates may be used for
screening for specific interactions with sequence specific targets
or probes.
In addition, the availability of substrates having the entire
repertoire of possible sequences of a defined length opens up the
possibility of sequencing by hybridization. This sequence may be de
novo determination of an unknown sequence, particularly of nucleic
acid, verification of a sequence determined by another method, or
an investigation of changes in a previously sequenced gene,
locating and identifying specific changes. For example, often Maxam
and Gilbert sequencing techniques are applied to sequences which
have been determined by Sanger and Coulson. Each of those
sequencing technologies have problems with resolving particular
types of sequences. Sequencing by hybridization may serve as a
third and independent method for verifying other sequencing
techniques. See, e.g., (1988) Science 242:1245.
In addition, the ability to provide a large repertoire of
particular sequences allows use of short subsequences and
hybridization as a means to fingerprint a sample. This may be used
in a nucleic acid, as well as other polymer embodiments. For
example, fingerprinting to a high degree of specificity of sequence
matching may be used for identifying highly similar samples, e.g.,
those exhibiting high homology to the selected probes. This may
provide a means for determining classifications of particular
sequences. This should allow determination of whether particular
genomes of bacteria, phage, or even higher cells might be related
to one another.
In addition, fingerprinting may be used to identify an individual
source of biological sample. See, e.g., Lander, E. (1989) Nature,
339:501-505, and references therein. For example, a DNA fingerprint
may be used to determine whether a genetic sample arose from
another individual. This would be particularly useful in various
sorts of forensic tests to determine, e.g., paternity or sources of
blood samples. Significant detail on the particulars of genetic
fingerprinting for identification purposes are described in, e.g.,
Morris et al. (1989) "Biostatistical evolution of evidence from
continuous allele frequency distribution DNA probes in reference to
disputed paternity of identity," J. Forensic Science 34:1311-1317;
and Neufeld et al. (1990) Scientific American 262:46-53; each of
which is hereby incorporated herein by reference.
In another embodiment, a fingerprinting-like procedure may be used
for classifying cell types by analyzing a pattern of specific
nucleic acids present in the cell. A series of antibodies may be
used to identify cell markers, e.g., proteins, usually on the cell
surface, but intracellular markers may also be used. Antigens which
are extracellularly expressed are preferred so cell lysis is
unnecessary in the screening, but intracellular markers may also be
useful. The markers will usually be proteins, but may be nucleic
acids, lipids, metabolites, carbohydrates, or other cellular
components. See, e.g., Winkelgren, I. (1990) Science News
136:234-237, which indicates extracellular DNA may be common, and
suggesting that such might be characteristic of cell types, stage,
or physiology. This may also be useful in defining the temporal
stage of development of cells, e.g., stem cells or other cells
which undergo temporal changes in development. For example, the
stage of a cell, or group of cells, may be tested or defined by
isolating a sample of mRNA from the population and testing to see
what sequences are present in messenger populations. Direct
samples, or amplified samples, may be used. Where particular mRNA
or other nucleic acid sequences may be characteristic of or shown
to be characteristic of particular developmental stages,
physiological states, or other conditions, this fingerprinting
method may define them. Similar sorts of fingerprinting may be used
for determining T-cell classes or perhaps even to generate
classification schemes for such proteins as major
histocompatibility complex antigens. Thus, the ability to make
these substrates allows both the generation of reagents which will
be used for defining subclasses or classes of cells or other
biological materials, but also provides the mechanisms for
selecting those cells which may be found in defined population
groups.
In addition to cell classification defined by such a combination of
properties, typically expression of extracellular antigens, the
present invention also provides the means for isolating homogeneous
population of cells. Once the antigenic determinants which define a
cell class have been identified, these antigens may be used in a
sequential selection process to isolate only those cells which
exhibit the combination of defining structural properties.
The present invention may also be used for mapping sequences within
a larger segment. This may be performed by at least two methods,
particularly in reference to nucleic acids. Often, enormous
segments of DNA are subcloned into a large plurality of
subsequences. Ordering these subsequences may be important in
determining the overlaps of sequences upon nucleotide
determinations. Mapping may be performed by immobilizing
particularly large segments onto a matrix using the VLSIPS.TM.
Technology. Alternatively, sequences may be ordered by virtue of
subsequences shared by overlapping segments. See, e.g., Craig et
al. (1990) Nuc. Acids Res. 18:2653-2660; Michiels et al. (1987)
CABIOS 3:203-210; and Olson et al. (1986) Proc. Natl. Acad. Sci.
USA 83:7826-7830.
B. Important Parameters
The extent of specific interaction between reagents immobilized to
the VLSIPS.TM. Technology substrate and another sequence specific
reagent may be modified by the conditions of the interaction.
Sequencing embodiments typically require high fidelity
hybridization and the ability to discriminate perfect matching from
imperfect matching. Fingerprinting and mapping embodiments may be
performed using less stringent conditions, depending upon the
circumstances.
For example, the specificity of antibody/antigen interaction may
depend upon such parameters as pH, salt concentration, ionic
composition, solvent composition, detergent composition and
concentration, and chaotropic agent concentration. See, e.g.,
Harlow and Lane (1988) Antibodies: A Laboratory Manual, Cold Spring
Harbor Press, New York. By careful control of these parameters, the
affinity of binding may be mapped across different sequences.
In a nucleic acid hybridization embodiment, the specificity and
kinetics of hybridization have been described in detail by, e.g.,
Wetmur and Davidson (1968) J. Mol. Biol., 31:349-370, Britten and
Kohne (1968) Science 161:529-530, and Kanehisa, (1984) Nuc. Acids
Res. 12:203-213, each of which is hereby incorporated herein by
reference. Parameters which are well known to affect specificity
and kinetics of reaction include salt conditions, ionic composition
of the solvent, hybridization temperature, length of
oligonucleotide matching sequences, guanine and cytosine (GC)
content, presence of hybridization accelerators, pH, specific bases
found in the matching sequences, solvent conditions, and addition
of organic solvents.
In particular, the salt conditions required for driving highly
mismatched sequences to completion typically include a high salt
concentration. The typical salt used is sodium chloride (NaCl),
however, other ionic salts may be utilized, e.g., KCl. Depending on
the desired stringency hybridization, the salt concentration will
often be less than about 3 molar, more often less than 2.5 molar,
usually less than about 2 molar, and more usually less than about
1.5 molar. For applications directed towards higher stringency
matching, the salt concentrations would typically be lower.
Ordinary high stringency conditions will utilize salt concentration
of less than about 1 molar, more often less then about 750
millimolar, usually less than about 500 millimolar, and may be as
low as about 250 or 150 millimolar.
The kinetics of hybridization and the stringency of hybridization
both depend upon the temperature at which the hybridization is
performed and the temperature at which the washing steps are
performed. Temperatures at which steps for low stringency
hybridization are desired would typically be lower temperatures,
e.g., ordinarily at least about 15.degree. C., more ordinarily at
least about 20.degree. C., usually at least about 25.degree. C.,
and more usually at least about 30.degree. C. For those
applications requiring high stringency hybridization, or fidelity
of hybridization and sequence matching, temperatures at which
hybridization and washing steps are performed would typically be
high. For example, temperatures in excess of about 35.degree. C.
would often be used, more often in excess of about 40.degree. C.,
usually at least about 45.degree. C., and occasionally even
temperatures as high as about 50.degree. C. or 60.degree. C. or
more. Of course, the hybridization of oligonucleotides may be
disrupted by even higher temperatures. Thus, for stripping of
targets from substrates, as discussed below, temperatures as high
as 80.degree. C., or even higher may be used.
The base composition of the specific oligonucleotides involved in
hybridization affects the temperature of melting, and the stability
of hybridization as discussed in the above references. However, the
bias of GC rich sequences to hybridize faster and retain stability
at higher temperatures can be compensated for by the inclusion in
the hybridization incubation or wash steps of various buffers.
Sample buffers which accomplish this result include the
triethly-and trimethyl ammonium buffers. See, e.g., Wood et al.
(1987) Proc. Natl. Acad. Sci. USA, 82:1585-1588, and Khrapko, K. et
al. (1989) FEBS Letters 256:118-122.
The rate of hybridization can also be affected by the inclusion of
particular hybridization accelerators. These hybridization
accelerators include the volume exclusion agents characterized by
dextran sulfate, or polyethylene glycol (PEG). Dextran sulfate is
typically included at a concentration of between 1% and 40% by
weight. The actual concentration selected depends upon the
application, but typically a faster hybridization is desired in
which the concentration is optimized for the system in question.
Dextran sulfate is often included at a concentration of between
0.5% and 2% by weight or dextran sulfate at a concentration between
about 0.5% and 5%. Alternatively, proteins which accelerate
hybridization may be added, e.g., the recA protein found in E. coli
or other homologous proteins.
With respect to those embodiments where specific reagents are not
oligonucleotides, the conditions of specific interaction would
depend on the affinity of binding between the specific reagent and
its target. Typically parameters which would be of particular
importance would be pH, salt concentration anion and cation
compositions, buffer concentration, organic solvent inclusion,
detergent concentration, and inclusion of such reagents such as
chaotropic agents. In particular, the affinity of binding may be
tested over a variety of conditions by multiple washes and repeat
scans or by using reagents with differences in binding affinity to
determine which reagents bind or do not bind under the selected
binding and washing conditions. The spectrum of binding affinities
may provide an additional dimension of information which may be
very useful in identification purposes and mapping.
Of course, the specific hybridization conditions will be selected
to correspond to a discriminatory condition which provides a
positive signal where desired but fails to show a positive signal
at affinities where interaction is not desired. This may be
determined by a number of titration steps or with a number of
controls which will be run during the hybridization and/or washing
steps to determine at what point the hybridization conditions have
reached the stage of desired specificity.
IX. DETECTION METHODS
Methods for detection depend upon the label selected. The criteria
for selecting an appropriate label are discussed below, however, a
fluorescent label is preferred because of its extreme sensitivity
and simplicity. Standard labeling procedures are used to determine
the positions where interactions between a sequence and a reagent
take place. For example, if a target sequence is labeled and
exposed to a matrix of different probes, only those locations where
probes do interact with the target will exhibit any signal.
Alternatively, other methods may be used to scan the matrix to
determine where interaction takes place. Of course, the spectrum of
interactions may be determined in a temporal manner by repeated
scans of interactions which occur at each of a multiplicity of
conditions. However, instead of testing each individual interaction
separately, a multiplicity of sequence interactions may be
simultaneously determined on a matrix.
A. Labeling Techniques
The target polynucleotide may be labeled by any of a number of
convenient detectable markers. A fluorescent label is preferred
because it provides a very strong signal with low background. It is
also optically detectable at high resolution and sensitivity
through a quick scanning procedure. Other potential labeling
moieties include, radioisotopes, chemiluminescent compounds,
labeled binding proteins, heavy metal atoms, spectroscopic markers,
magnetic labels, and linked enzymes.
Another method for labeling may bypass any label of the target
sequence. The target may be exposed to the probes, and a double
strand hybrid is formed at those positions only. Addition of a
double strand specific reagent will detect where hybridization
takes place. An intercalative dye such as ethidium bromide may be
used as long as the probes themselves do not fold back on
themselves to a significant extent forming hairpin loops. See,
e.g., Sheldon et al. (1986) U.S. Pat. No. 4,582,789. However, the
length of the hairpin loops in short oligonucleotide probes would
typically be insufficient to form a stable duplex.
In another embodiment, different targets may be simultaneously
sequenced where each target has a different label. For instance,
one target could have a green fluorescent label and a second target
could have a red fluorescent label. The scanning step will
distinguish sites of binding of the red label from those binding
the green fluorescent label. Each sequence can be analyzed
independently from one another.
Suitable chromogens will include molecules and compounds which
absorb light in a distinctive range of wavelengths so that a color
may be observed, or emit light when irradiated with radiation of a
particular wave length or wave length range, e.g., fluorescers.
Biliproteins, e.g., phycoerythrin, may also serve as labels.
A wide variety of suitable dyes are available, being primarily
chosen to provide an intense color with minimal absorption by their
surroundings. Illustrative dye types include quinoline dyes,
triarylmethane dyes, acridine dyes, alizarine dyes, phthaleins,
insect dyes, azo dyes, anthraquinoid dyes, cyanine dyes,
phenazathionium dyes, and phenazoxonium dyes.
A wide variety of fluorescers may be employed either by themselves
or in conjunction with quencher molecules. Fluorescers of interest
fall into a variety of categories having certain primary
functionalities. These primary functionalities include 1- and
2-aminonaphthalene, p,p'-diaminostilbenes, pyrenes, quaternary
phenanthridine salts, 9-aminoacridines, p,p'-diaminobenzophenone
imines, anthracenes, oxacarbocyanine, merocyanine,
3-aminoequilenin, perylene, bisbenzoxazole, bis-p-oxazolyl benzene,
1,2-benzophenazin, retinol, bis-3-aminopyridinium salts,
hellebrigenin, tetracycline, sterophenol,
benzimidzaolylphenylamine, 2-oxo-3-chromen, indole, xanthen,
7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin,
porphyrins, triarylmethanes and flavin. Individual fluorescent
compounds which have functionalities for linking or which can be
modified to incorporate such functionalities include, e.g., dansyl
chloride; fluoresceins such as 3,6-dihydroxy-9-phenylxanthhydrol;
rhodamineisothiocyanate; N-phenyl 1-amino-8-sulfonatonaphthalene;
N-phenyl 2-amino-6-sulfonatonaphthalene;
4-acetamido-4-isothiocyanato-stilbene-2,2'-disulfonic acid;
pyrene-3-sulfonic acid; 2-toluidinonaphthalene-6-sulfonate;
N-phenyl, N-methyl 2-aminoaphthalene-6-sulfonate; ethidium bromide;
stebrine; auromine-0,2-(9'-anthroyl)palmitate; dansyl
phosphatidylethanolamine; N,N'-dioctadecyl oxacarbocyanine;
N,N'-dihexyl oxacarbocyanine; merocyanine, 4-(3'pyrenyl)butyrate;
d-3-aminodesoxy-equilenin; 12-(9'-anthroyl)stearate;
2-methylanthracene; 9-vinylanthracene;
2,2'-(vinylene-p-phenylene)bisbenzoxazole;
p-bis[2-(4-methyl-5-phenyl-oxazolyl)]benzene;
6-dimethylamino-1,2-benzophenazin; retinol; bis
(3'-aminopyridinium) 1,10-decandiyl diiodide;
sulfonaphthylhydrazone of hellibrienin; chlorotetracycline;
N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;
N-[p-(2-benzimidazolyl)-phenyl]maleimide;
N-(4-fluoranthyl)maleimide; bis(homovanillic acid); resazarin;
4-chloro-7-nitro-2,1,3-benzooxadiazole; merocyanine 540; resorufin;
rose bengal; and 2,4-diphenyl-3(2H)-furanone.
Desirably, fluorescers should absorb light above about 300 nm,
preferably about 350 nm, and more preferably above about 400 nm,
usually emitting at wavelengths greater than about 10 nm higher
than the wavelength of the light absorbed. It should be noted that
the absorption and emission characteristics of the bound dye may
differ from the unbound dye. Therefore, when referring to the
various wavelength ranges and characteristics of the dyes, it is
intended to indicate the dyes as employed and not the dye which is
unconjugated and characterized in an arbitrary solvent.
Fluorescers are generally preferred because by irradiating a
fluorescer with light, one can obtain a plurality of emissions.
Thus, a single label can provide for a plurality of measurable
events.
Detectable signal may also be provided by chemiluminescent and
bioluminescent sources. Chemiluminescent sources include a compound
which becomes electronically excited by a chemical reaction and may
then emit light which serves as the detectible signal or donates
energy to a fluorescent acceptor. A diverse number of families of
compounds have been found to provide chemiluminescence under a
variety of conditions. One family of compounds is
2,3-dihydro-1,-4-phthalazinedione. The most popular compound is
luminol, which is the 5-amino compound. Other members of the family
include the 5-amino-6,7,8-trimethoxy- and the dimethylamino[ca]benz
analog. These compounds can be made to luminesce with alkaline
hydrogen peroxide or calcium hypochlorite and base. Another family
of compounds is the 2,4,5-triphenylimidazoles, with lophine as the
common name for the parent product. Chemiluminescent analogs
include para-dimethylamino and -methoxy substituents.
Chemiluminescence may also be obtained with oxalates, usually
oxalyl active esters, e.g., p-nitrophenyl and a peroxide, e.g.,
hydrogen peroxide, under basic conditions. Alternatively,
luciferins may be used in conjunction with luciferase or lucigenins
to provide bioluminescence.
Spin labels are provided by reporter molecules with an unpaired
electron spin which can be detected by electron spin resonance
(ESR) spectroscopy. Exemplary spin labels include organic free
radicals, transitional metal complexes, particularly vanadium,
copper, iron, and manganese, and the like. Exemplary spin labels
include nitroxide free radicals.
B. Scanning System
With the automated detection apparatus, the correlation of specific
positional labeling is converted to the presence on the target of
sequences for which the reagents have specificity of interaction.
Thus, the positional information is directly converted to a
database indicating what sequence interactions have occurred. For
example, in a nucleic acid hybridization application, the sequences
which have interacted between the substrate matrix and the target
molecule can be directly listed from the positional information.
The detection system used is described in Pirrung et al. (1992)
U.S. Pat. No. 5,143,854; and Ser. No. 07/624,120, now abandoned.
Although the detection described therein is a fluorescence
detector, the detector may be replaced by a spectroscopic or other
detector. The scanning system may make use of a moving detector
relative to a fixed substrate, a fixed detector with a moving
substrate, or a combination. Alternatively, mirrors or other
apparatus can be used to transfer the signal directly to the
detector. See, e.g, Ser. No. 07/624,120, now abandoned, which is
hereby incorporated herein by reference.
The detection method will typically also incorporate some signal
processing to determine whether the signal at a particular matrix
position is a true positive or may be a spurious signal. For
example, a signal from a region which has actual positive signal
may tend to spread over and provide a positive signal in an
adjacent region which actually should not have one. This may occur,
e.g., where the scanning system is not properly discriminating with
sufficiently high resolution in its pixel density to separate the
two regions. Thus, the signal over the spatial region may be
evaluated pixel by pixel to determine the locations and the actual
extent of positive signal. A true positive signal should, in
theory, show a uniform signal at each pixel location. Thus,
processing by plotting number of pixels with actual signal
intensity should have a clearly uniform signal intensity. Regions
where the signal intensities show a fairly wide dispersion, may be
particularly suspect and the scanning system may be programmed to
more carefully scan those positions.
In another embodiment, as the sequence of a target is determined at
a particular location, the overlap for the sequence would
necessarily have a known sequence. Thus, the system can compare the
possibilities for the next adjacent position and look at these in
comparison with each other. Typically, only one of the possible
adjacent sequences should give a positive signal and the system
might be programmed to compare each of these possibilities and
select that one which gives a strong positive. In this way, the
system can also simultaneously provide some means of measuring the
reliability of the determination by indicating what the average
signal to background ratio actually is.
More sophisticated signal processing techniques can be applied to
the initial determination of whether a positive signal exists or
not. See, e.g., Ser. No. 07/624,120, now abandoned.
From a listing of those sequences which interact, data analysis may
be performed on a series of sequences. For example, in a nucleic
acid sequence application, each of the sequences may be analyzed
for their overlap regions and the original target sequence may be
reconstructed from the collection of specific subsequences obtained
therein. Other sorts of analyses for different applications may
also be performed, and because the scanning system directly
interfaces with a computer the information need not be transferred
manually. This provides for the ability to handle large amounts of
data with very little human intervention. This, of course, provides
significant advantages over manual manipulations. Increased
throughput and reproducibility is thereby provided by the
automation of a vast majority of steps in any of these
applications.
XI. DATA ANALYSIS
A. General
Data analysis will typically involve aligning the proper sequences
with their overlaps to determine the target sequence. Although the
target "sequence" may not specifically correspond to any specific
molecule, especially where the target sequence is broken and
fragmented in the sequencing process, the sequence corresponds to a
contiguous sequence of the subfragments.
The data analysis can be performed by a computer using an
appropriate program. See, e.g., Drmanac, R. et al. (1989) Genomics
4:114-128; and a commercially available analysis program available
from the Genetic Engineering Center, P.O. Box 794, 11000 Belgrade,
Yugoslavia. Although the specific manipulations necessary to
reassemble the target sequence from fragments may take many forms,
one embodiment uses a sorting program to sort all of the
subsequences using a defined hierarchy. The hierarchy need not
necessarily correspond to any physical hierarchy, but provides a
means to determine, in order, which subfragments have actually been
found in the target sequence. In this manner, overlaps can be
checked and found directly rather than having to search throughout
the entire set after each selection process. For example, where the
oligonucleotide probes are 10-mers, the first 9 positions can be
sorted. A particular subsequence can be selected as in the
examples, to determine where the process starts. As analogous to
the theoretical example provided above, the sorting procedure
provides the ability to immediately find the position of the
subsequence which contains the first 9 positions and can compare
whether there exists more than 1 subsequence during the first 9
positions. In fact, the computer can easily generate all of the
possible target sequences which contain given combination of
subsequences. Typically there will be only one, but in various
situations, there will be more.
An exemplary flow chart for a sequencing program is provided in
FIG. 2. In general terms, the program provides for automated
scanning of the substrate to determine-the positions of probe and
target interaction. Simple processing of the intensity of the
signal may be incorporated to filter out clearly spurious signals.
The positions with positive interaction are correlated with the
sequence specificity of specific matrix positions, to generate the
set of matching subsequences. This information is further
correlated with other target sequence information, e.g.,
restriction fragment analysis. The sequences are then aligned using
overlap data, thereby leading to possible corresponding target
sequences which will, optimally, correspond to a single target
sequence.
B. Hardware
A variety of computer systems may be used to run a sequencing
program. The program may be written to provide both the detecting
and scanning steps together and will typically be dedicated to a
particular scanning apparatus. However, the components and
functional steps may be separated and the scanning system may
provide an output, e.g., through tape or an electronic connection
into a separate computer which separately runs the sequencing
analysis program. The computer may be any of a number of machines
provided by standard computer manufacturers, e.g., IBM compatible
machines, Apple.TM. machines, VAX machines, and others, which may
often use a UNIX.TM. operating system. Of course, the hardware used
to run the analysis program will typically determine what
programming language would be used.
C. Software
Software would be easily developed by a person of ordinary skill in
the programming art, following the flow chart provided, or based
upon the input provided and the desired result.
Of course, an exemplary embodiment is a polynucleotide sequence
system. However, the theoretical and mathematical manipulations
necessary for data analysis of other linear molecules, such as
polypeptides, carbohydrates, and various other polymers are
conceptually similar. Simple branching polymers will usually also
be sequencable using similar technology. However, where there is
branching, it may be desired that additional recognition reagents
be used to determine the nature and location of branches. This can
easily be provided by use of appropriate specific reagents which
would be generated by methods similar to those used to produce
specific reagents for linear polymers.
XII. SUBSTRATE REUSE
Where a substrate is made with specific reagents that are
relatively insensitive to the handling and processing steps
involved in a single cycle of use, the substrate may often be
reused. The target molecules are usually stripped off of the solid
phase specific recognition molecules. Of course, it is preferred
that the manipulations and conditions be selected as to be mild and
to not affect the substrate. For example, if a substrate is acid
labile, a neutral pH would be preferred in all handling steps.
Similar sensitivities would be carefully respected where recycling
is desired.
A. Removal of Label
Typically for a recycling, the previously attached specific
interaction would be disrupted and removed. This will typically
involve exposing the substrate to conditions under which the
interaction between probe and target is disrupted. Alternatively,
it may be exposed to conditions where the target is destroyed. For
example, where the probes are oligonucleotides and the target is a
polynucleotide, a heating and low salt wash will often be
sufficient to disrupt the interactions. Additional reagents may be
added such as detergents, and organic or inorganic solvents which
disrupt the interaction between the specific reagents and target.
In an embodiment where the specific reagents are antibodies, the
substrate may be exposed to a gentle detergent which will denature
the specific binding between the antibody and its target. The
conditions are selected to avoid severe disruption or destruction
of the structure of the antibody and to maintain the specificity of
the antibody binding site. Conditions with specific pH, detergent
concentration, salt concentration, ionic concentration, and other
parameters may be selected which disrupt the specific
interactions.
B. Storage and Preservation
As indicated above, the matrix will typically be maintained under
conditions where the matrix itself and the linkages and specific
reagents are preserved. Various specific preservatives may be added
which prevent degradation. For example, if the reagents are acid or
base labile, a neutral pH buffer will typically be added. It is
also desired to avoid destruction of the matrix by growth of
organisms which may destroy organic reagents attached thereto. For
this reason, a preservative such as cyanide or azide may be added.
However, the chemical preservative should also be selected to
preserve the chemical nature of the linkages and other components
of the substrate. Typically, a detergent may also be included.
C. Processes to Avoid Degradation of Oligomers
In particular, a substrate comprising a large number of oligomers
will be treated in a fashion which is known to maintain the quality
and integrity of oligonucleotides. These include storing the
substrate in a carefully controlled environment under conditions of
lower temperature, cation depletion (EDTA and EGTA), sterile
conditions, and inert argon or nitrogen atmosphere.
XIII. INTEGRATED SEQUENCING STRATEGY
A. Initial Mapping Strategy
As indicated above, although the VLSIPS.TM. technology may be
applied to sequencing embodiments, it is often useful to integrate
other concepts to simplify the sequencing. For example, nucleic
acids may be easily sequenced by careful selection of the vectors
and hosts used for amplifying and generating the specific target
sequences. For example, it may be desired to use specific vectors
which have been designed to interact most efficiently with the
VLSIPS substrate. This is also important in fingerprinting and
mapping strategies. For example, vectors may be carefully selected
having particular complementary sequences which are designed to
attach to a genetic or specific oligomer on the substrate. This is
also applicable to situations where it is desired to target
particular sequences to specific locations on the matrix.
In one embodiment, unnatural oligomers may be used to target
natural probes to specific locations on the VLSIPS substrate. In
addition, particular probes may be generated for the mapping
embodiment which are designed to have specific combinations of
characteristics. For example, the construction of a mapping
substrate may depend upon use of another automated apparatus which
takes clones isolated from a chromosome walk and attaches them
individually or in bulk to the VLSIPS substrate.
In another embodiment, a variety of specific vectors having known
and particular "targeting" sequences adjacent to the cloning sites
may be individually used to clone a selected probe, and the
isolated probe will then be targetable to a site on the VLSIPS
substrate with a sequence complementary to the "target"
sequence.
B. Selection of Smaller Clones
In the fingerprinting and mapping embodiments, the selection of
probes may be very important. Significant mathematical analysis may
be applied to determine which specific sequences should be used as
those probes. Of course, for fingerprinting use, these sequences
would be most desired that show significant heterogeneity across
the human population. Selection of the specific sequences which
would most favorably be utilized will tend to be single copy
sequences within the genome.
Various hybridization selection procedures may be applied to select
sequences which tend not to be repeated within a genome, and thus
would tend to be conserved across individuals. For example,
hybridization selections may be made for non-repetitive and single
copy sequences. See, e.g., Britten and Kohne (1968) "Repeated
Sequences in DNA," Science 161:529-540. On the other hand, it may
be desired under certain circumstances to use repeated sequences.
For example, where a fingerprint may be used to identify or
distinguish different species, or where repetitive sequences may be
diagnostic of specific species, repetitive sequences may be desired
for inclusion in the fingerprinting probes. In either case, the
sequencing capability will greatly assist in the selection of
appropriate sequences to be used as probes.
Also as indicated above, various means for constructing an
appropriate substrate may involve either mechanical or automated
procedures. The standard VLSIPS automated procedure involves
synthesizing oligonucleotides or short polymers directly on the
substrate. In various other embodiments, it is possible to attach
separately synthesized reagents onto the matrix in an ordered
array. Other circumstances may lend themselves to transfer a
pattern from a petri plate onto a solid substrate. Also, there are
methods for site specifically directing collections of reagents to
specific locations using unnatural nucleotides or equivalent sorts
of targeting molecules.
While a brute force manual transfer process may be utilized
sequentially for attaching various samples to successive positions,
instrumentation for automating such procedures may also be devised.
The automated system for performing such would preferably be
relatively easily designed and conceptually easily understood.
XIV. COMMERCIAL APPLICATIONS
A. Sequencing
As indicated above, sequencing may be performed either de novo or
as a verification of another sequencing method. The present
hybridization technology provides the ability to sequence nucleic
acids and polynucleotides de novo, or as a means to verify either
the Maxam and Gilbert chemical sequencing technique or Sanger and
Coulson dideoxy- sequencing techniques. The hybridization method is
useful to verify sequencing determined by any other sequencing
technique and to closely compare two similar sequences, e.g., to
identify and locate sequence differences.
Besides polynucleotide sequencing, the present invention also
provides means for sequencing other polymers. This includes
polypeptides, carbohydrates, synthetic organic polymers, and other
polymers. Again, the sequencing may be either verification or de
novo.
Of course, sequencing can be very important in many different sorts
of environments. For example, it will be useful in determining the
genetic sequence of particular markers in various individuals. In
addition, polymers may be used as markers or for information
containing molecules to encode information. For example, a short
polynucleotide sequence may be included in large bulk production
samples indicating the manufacturer, date, and location of
manufacture of a product. For example, various drugs may be encoded
with this information with a small number of molecules in a batch.
For example, a pill may have somewhere from 10 to 100 to 1,000 or
more very short and small molecules encoding this information. When
necessary, this information may be decoded from a sample of the
material using a polymerase chain reaction (PCR) or other
amplification method. This encoding system may be used to provide
the origin of large bulky samples without significantly affecting
the properties of those samples. For example, chemical samples may
also be encoded by this method thereby providing means for
identifying the source and manufacturing details of lots. The
origin of bulk hydrocarbon samples may be encoded. Production lots
of organic compounds such as benzene or plastics may be encoded
with a short molecule polymer. Food stuffs may also be encoded
using similar marking molecules. Even toxic waste samples can be
encoded determining the source or origin. In this way, proper
disposal can be traced or more easily enforced.
Similar sorts of encoding may be provided by fingerprinting-type
analysis. Whether the resolution is absolute or less so, the
concept of coding information on molecules such as nucleic acids,
which can be amplified and later decoded, may be a very useful and
important application.
This technology also provides the ability to include markers for
origins of biological materials. For example, a patented animal
line may be transformed with a particular unnatural sequence which
can be traced back to its origin. With a selection of multiple
markers, the likelihood could be negligible that a combination of
markers would have independently arisen from a source other than
the patented or specifically protected source. This technique may
provide a means for tracing the actual origin of particular
biological materials. Bacteria, plants, and animals will be subject
to marking by such encoding sequences.
B. Fingerprinting
As indicated above, fingerprinting technology may also be used for
data encryption. Moreover, fingerprinting allows for significant
identification of particular individuals. Where the fingerprinting
technology is standardized, and used for identification of large
numbers of people, related equipment and peripheral processing will
be developed to accompany the underlying technology. For example,
specific equipment may be developed for automatically taking a
biological sample and generating or amplifying the information
molecules within the sample to be used in fingerprinting analysis.
Moreover, the fingerprinting substrate may be mass produced using
particular types of automatic equipment. Synthetic equipment may
produce the entire matrix simultaneously by stepwise synthetic
methods as provided by the VLSIPS.TM. technology. The attachment of
specific probes onto a substrate may also be automated, e.g.,
making use of the caged biotin technology. See, e.g., Barrett et
al. (1993) U.S. Pat. No. 5,252,743. As indicated above, there are
automated methods for actually generating the matrix and substrate
with distinct sequence reagents positionally located at each of the
matrix positions. Where such reagents are, e.g., unnatural amino
acids, a targeting function may be utilized which does not
interfere with a natural nucleotide functionality.
In addition, peripheral processing may be important and may be
dedicated to this specific application. Thus, automated equipment
for producing the substrates may be designed, or particular systems
which take in a biological sample and output either a computer
readout or an encoded instrument, e.g., a card or document which
indicates the information and can provide that information to
others. An identification having a short magnetic strip with a few
million bits may be used to provide individual identification and
important medical information useful in a medical emergency.
In fact, data banks may be set up to correlate all of this
information of fingerprinting with medical information. This may
allow for the determination of correlations between various medical
problems and specific DNA sequences. By collating large populations
of medical records with genetic information, genetic propensities
and genetic susceptibilities to particular medical conditions may
be developed. Moreover, with standardization of substrates, the
micro encoding data may be also standardized to reproduce the
information from a centralized data bank or on an encoding device
carried on an individual person. On the other hand, if the
fingerprinting procedure is sufficiently quick and routine, every
hospital may routinely perform a fingerprinting operation and from
that determine many important medical parameters for an
individual.
In particular industries, the VLSIPS sequencing, fingerprinting, or
mapping technology will be particularly appropriate. As mentioned
above, agricultural livestock suppliers may be able to encode and
determine whether their particular strains are being used by
others. By incorporating particular markers into their genetic
stocks, the markers will indicate origin of genetic material. This
is applicable to seed producers, livestock producers, and other
suppliers of medical or agricultural biological materials.
This may also be useful in identifying individual animals or
plants. For example, these markers may be useful in determining
whether certain fish return to their original breeding grounds,
whether sea turtles always return to their original birthplaces, or
to determine the migration patterns and viability of populations of
particular endangered species. It would also provide means for
tracking the sources of particular animal products. For example, it
might be useful for determining the origins of controlled animal
substances such as elephant ivory or particular bird populations
whose importation or exportation is controlled.
As indicated above, polymers may be used to encode important
information on source and batch and supplier. This is described in
greater detail, e.g., "Applications of PCR to industrial problems,"
(1990) in Chemical and Engineering News 68:145, which is hereby
incorporated herein by reference. In fact, the synthetic method can
be applied to the storage of enormous amounts of information. Small
substrates may encode enormous amounts of information, and its
recovery will make use of the inherent replication capacity. For
example, on regions of 10 .mu.m.times.10 .mu.m, 1 cm.sup.2 has
10.sup.6 regions. In theory, the entire human genome could be
attached in 1000 nucleotide segments on a 3 cm.sup.2 surface.
Genomes of endangered species may be stored on these
substrates.
Fingerprinting may also be used for genetic tracing or for
identifying individuals for forensic science purposes. See, e.g.,
Morris, J. et al. (1989) "Biostatistical Evaluation of Evidence
From Continuous Allele Frequency Distribution DNA Probes in
Reference to Disputed Paternity and Identity," J. Forensic Science
34:1311-1317, and references provided therein; each of which is
hereby incorporated herein by reference.
In addition, the high resolution fingerprinting allows the
distinguishability to high resolution of particular samples. As
indicated above, new cell classifications may be defined based on
combinations of a large number of properties. Similar applications
will be found in distinguishing different species of animals or
plants. In fact, microbial identification may become dependent on
characterization of the genetic content. Tumors or other cells
exhibiting abnormal physiology will be detectable by use of the
present invention. Also, knowing the genetic fingerprint of a
microorganism may provide very useful information on how to treat
an infection by such organism.
Modifications of the fingerprint embodiments may be used to
diagnose the condition of the organism. For example, a blood sample
is presently used for diagnosing any of a number of different
physiological conditions. A multi-dimensional fingerprinting method
made available by the present invention could become a routine
means for diagnosing an enormous number of physiological features
simultaneously. This may revolutionize the practice of medicine in
providing information on an enormous number of parameters together
at one time. In another way, the genetic predisposition may also
revolutionize the practice of medicine providing a physician with
the ability to predict the likelihood of particular medical
conditions arising at any particular moment. It also provides the
ability to apply preventive medicine.
The present invention might also find application in use for
screening new drugs and new reagents which may be very important in
medical diagnosis or other applications. For example, a description
of generating a population of monoclonal antibodies with defined
specificities may be very useful for producing various drugs or
diagnostic reagents.
Also available are kits with the reagents useful for performing
sequencing, fingerprinting, and mapping procedures. The kits will
have various compartments with the desired necessary reagents,
e.g., substrate, labeling reagents for target samples, buffers, and
other useful accompanying products.
C. Mapping
The present invention also provides the means for mapping sequences
within enormous stretches of sequence. For example, nucleotide
sequences may be mapped within enormous chromosome size sequence
maps. For example, it would be possible to map a chromosomal
location within the chromosome which contains hundreds of millions
of nucleotide base pairs. In addition, the mapping and
fingerprinting embodiments allow for testing of chromosomal
translocations, one of the standard problems for which
amniocentesis is performed.
Thus, the present invention provides a powerful tool and the means
for performing sequencing, fingerprinting, and mapping functions on
polymers. Although most easily and directly applicable to
polynucleotides, polypeptides, carbohydrates, and other sorts of
molecules can be advantageously utilized using the present
technology.
The present invention will be better understood by reference to the
following illustrative examples. The following examples are offered
by way of illustration and not by way of limitation.
EXPERIMENTAL
I. Sequencing
A. polynucleotide
B. polypeptide
C. short peptide
1. Herz antibody identification
II. Fingerprinting
A. polynucleotide fingerprint
B. peptide fingerprint
C. cell classification scheme
D. temporal development scheme
1. developmental antigens
2. developmental mRNA expression
E. diagnostic test
1. viral identification
2. bacterial identification
3. other microbiological identifications
4. allergy test (immobilized antigens)
F. individual (animal/plant) identification
1. genetic
2. immunological
G. genetic screen
1. test alleles with markers
2. amniocentesis
III. Mapping
A. positionally located clones (caged biotin)
1. short probes, long targets
2. long targets, short probes
B. positionally defined clones
IV. Conclusion
* * * * * * * * * * * * * * *
Relevant applications whose techniques are incorporated herein by
reference are Pirrung, et al., Ser. No. 07/362,901, filed Jun. 7,
1989, now abandoned; Pirrung et al. (1992) U.S. Pat. No. 5,143,854;
Barrett, et al., Ser. No. 07/435,316 filed Nov. 13, 1989, now
abandoned; Barrett, et al. (1993) U.S. Pat. No. 5,252,743; and
commonly assigned and simultaneously filed applications Ser. No.
07/624,120, now abandoned, and Ser. No. 07/626,730.
Also, additional relevant techniques are described, e.g., in
Sambrook, J., et al. (1989) Molecular Cloning: a Laboratory Manual,
2d Ed., vols 1-3, Cold Spring Harbor Press, New York; Greenstein
and Winitz (1961) Chemistry of the Amino Acids, Wiley and Sons, New
York; Bodzansky, M. (1988) Peptide Chemistry: a Practical Textbook,
Springer-Verlag, New York; Harlow and Lane (1988) Antibodies: A
Laboratory Manual, Cold Spring Harbor Press, New York; Glover, D.
(ed.) (1987) DNA Cloning: A Practical Approach, vols 1-3, IRL
Press, Oxford; Bishop and Rawlings (1987) Nucleic Acid and Protein
Sequence Analysis: A Practical Approach, IRL Press, Oxford; Hames
and Higgins (1985) Nucleic Acid Hybridisation: A Practical
Approach, IRL Press, Oxford; Wu et al. (1989) Recombinant DNA
Methodology, Academic Press, San Diego; Goding (1986) Monoclonal
Antibodies: Principles and Practice, (2d ed.), Academic Press, San
Diego; Finegold and Barron (1986) Bailey and Scott's Diagnostic
Microbiology, (7th ed.), Mosby Co., St. Louis; Collins et al.
(1989) Microbiological Methods, (6th ed.), Butterworth, London;
Chaplin and Kennedy (1986) Carbohydrate Analysis: A Practical
Approach, IRL Press, Oxford; Van Dyke (ed.) (1985) Bioluminescence
and Chemiluminescence: Instruments and Applications, vol 1, CRC
Press, Boca Rotan; and Ausubel et al. (ed.) (1990) Current
Protocols in Molecular Biology, Greene Publishing and
Wiley-Interscience, New York; each of which is hereby incorporated
herein by reference.
The following examples are provided to illustrate the efficacy of
the inventions herein. All operations were conducted at about
ambient temperatures and pressures unless indicated to the
contrary.
I. SEQUENCING
A. polynucleotide
1. HPLC of the photolysis of 5'-O-nitroveratryl-thymidine.
In order to determine the time for photolysis of 5'-O-nitroveratryl
thymidine to thymidine a 100 .mu.M solution of NV-Thym-OH
(5'-O-nitroveratryl thymidine) in dioxane was made and .sup..about.
200 .mu.l aliquots were irradiated (in a quartz cuvette 1
cm.times.2 mm) at 362.3 nm for 20 sec, 40 sec, 60 sec, 2 min, 5
min, 10 min, 15 min, and 20 min. The resulting irradiated mixtures
were then analyzed by HPLC using a Varian MicroPak SP column
(C.sub.18 analytical) at a flow rate of 1 ml/min and a solvent
system of 40% CH.sub.3 CN and 60% water. Thymidine has a retention
time of 1.2 min and NVO-Thym-OH has a retention time of 2.1 min. It
was seen that after 10 min of exposure the deprotection was
complete.
2. Preparation and Detection of Thymidine-Cytidine dimer (FITC)
The reaction is illustrated: ##STR18##
To an aminopropylated glass slide (standard VLSIPS.TM. Technology)
was added a mixture of the following:
12.2 mg of NVO-Thym-CO.sub.2 H (IX)
3.4 mg of HOBT (N-hydroxybenztriazal)
8.8 .mu.l DIEA (Diisopropylethylamine)
11.1 mg BOP reagent
2.5 ml DMF
After 2 h coupling time (standard VLSIPS) the plate was washed,
acetylated with acetic anhydride/pyridine, washed, dried, and
photolyzed in dioxane at 362 nm at 14 mW/cm.sup.2 for 10 in using a
500 .mu.m checkerboard mask. The slide was then taken and treated
with a mixture of the following:
107 mg of FMOC-amine modified C (III)
21 mg of tetrazole
1 ml anhydrous CH.sub.3 CN
After being treated for approximately 8 min, the slide was washed
off with CH.sub.3 CN, dried, and oxidized with I.sub.2 /H.sub.2
O/THF/lutidine for 1 min. The slide was again washed, dried, and
treated for 30 min with a 20% solution of DBU in DMF. After
thorough rinsing of the slide, it was next exposed to a FITC
solution (1 mM fluorescein isothiocyanate [FITC] in DMF) for 50
min, then washed, dried, and examined by fluorescence microscopy.
This reaction is illustrated: ##STR19##
Preparation and Detection of Thymidine-Cytidine dimer (Biotin)
An aminopropyl glass slide, was soaked in a solution of ethylene
oxide (20% in DMF) to generate a hydroxylated surface. The slide
was added to a mixture of the following:
32 mg of NVO-T-OCED (X)
11 mg of tetrazole
0.5 ml of anhydrous CH.sub.3 CN
After 8 min the plate was then rinsed with acetonitrile, then
oxidized with I.sub.2 /H.sub.2 O/THF/lutidine for 1 min, washed and
dried. The slide was then exposed to a 1:3 mixture of acetic
anhydride:pyridine for 1 h, then washed and dried. The substrate
was then photolyzed in dioxane at 362 nm at 14 mW/cm.sup.2 for 10
min using a 500 .mu.m checkerboard mask, dried, and then treated
with a mixture of the following:
65 mg of biotin modified C (IV)
11 mg of tetrazole
0.5 ml anhydrous CH.sub.3 CN
After 8 min the slide was washed with CH.sub.3 CN then oxidized
with I.sub.2 /H.sub.2 O/THF/lutidine for 1 min, washed, and then
dried. The slide was then soaked for 30 min in a PBS/0.05% Tween 20
buffer and the solution then shaken off. The slide was next treated
with FITC-labeled streptavidin at 10 .mu.g/ml in the same buffer
system for 30 min. After this time the streptavidin-buffer system
was rinsed off with fresh PBS/0.05% Tween 20 buffer and then the
slide was finally agitated in distilled water for about 1/2 h.
After drying, the slide was examined by fluorescence microscopy
(see FIG. 2 and FIG. 3).
4. Substrate Preparation
Before attachment of reactive groups it is preferred to clean the
substrate which is, in a preferred embodiment, a glass substrate
such as a microscope slide or cover slip. A roughened surface will
be useable but a plastic or other solid substrate is also
appropriate. According to one embodiment the slide is soaked in an
alkaline bath consisting of, e.g., 1 liter of 95% ethanol with 120
ml of water and 120 grams of sodium hydroxide for 12 hours. The
slides are washed with a buffer and under running water, allowed to
air dry, and rinsed with a solution of 95% ethanol.
The slides are then aminated with, e.g., aminopropyltriethoxysilane
for the purpose of attaching amino groups to the glass surface on
linker molecules, although other omega functionalized silanes could
also be used for this purpose. In one embodiment 0.1%
aminopropyltriethoxysilane is utilized, although solutions with
concentrations from 10.sup.-7 % to 10% may be used, with about
10.sup.-3 % to 2% preferred. A 0.1% mixture is prepared by adding
to 100 ml of a 95% ethanol/5% water mixture, 100 microliters
(.mu.l) of aminopropyltriethoxysilane. The mixture is agitated at
about ambient temperature on a rotary shaker for an appropriate
amount of time, e.g., about 5 minutes. 500 .mu.l of this mixture is
then applied to the surface of one side of each cleaned slide.
After 4 minutes or more, the slides are decanted of this solution
and thoroughly rinsed three times or more by dipping in 100%
ethanol.
After the slides dry, they are heated in a 110.degree.-120.degree.
C. vacuum oven for about 20 minutes, and then allowed to cure at
room temperature for about 12 hours in an argon environment. The
slides are then dipped into DMF (dimethylformamide) solution,
followed by a thorough washing with methylene chloride.
5. Linker Attachment, Blocking of Free Sites
The aminated surface of the slide is then exposed to about 500
.mu.l of, for example, a 30 millimolar (mM) solution of
NVOC-nucleotide- NHS (N-hydroxysuccinimide) in DMF for attachment
of a NVOC-nucleotide to each of the amino groups. See, e.g., SIGMA
Chemical Company for various nucleotide derivatives. The surface is
washed with, for example, DMF, methylene chloride, and ethanol.
Any unreacted aminopropyl silane on the surface, i.e., those amino
groups which have not had the NVOC-nucleotide attached, are now
capped with acetyl groups (to prevent further reaction) by exposure
to a 1:3 mixture of acetic anhydride in pyridine for 1 hour. Other
materials which may perform this residual capping function include
trifluoroacetic anhydride, formicacetic anhydride, or other
reactive acylating agents. Finally, the slides are washed again
with DMF, methylene chloride, and ethanol.
6. Synthesis of Eight Trimers of C and T
FIG. 2 illustrates a possible synthesis of the eight trimers of the
two-monomer set: cytosine and thymine (represented by C and T,
respectively). A glass slide bearing silane groups terminating in
6-nitroveratryloxycarboxamide (NVOC-NH) residues is prepared as a
substrate. Active esters (pentafluorophenyl, OBt, etc.) of cytosine
and thymine protected at the 5' hydroxyl group with NVOC are
prepared as reagents. While not pertinent to this example, if side
chain protecting groups are required for the monomer set, these
must not be photoreactive at the wavelength of light used to
protect the primary chain.
For a monomer set of size n, n.times.l cycles are required to
synthesize all possible sequences of length l. A cycle consists
of:
1. Irradiation through an appropriate mask to expose the 5'-OH
groups at the sites where the next residue is to be added, with
appropriate washes to remove the by-products of the
deprotection.
2. Addition of a single activated and protected (with the same
photochemically-removable group) monomer, which will react only at
the sites addressed in step 1, with appropriate washes to remove
the excess reagent from the surface.
The above cycle is repeated for each member of the monomer set
until each location on the surface has been extended by one residue
in one embodiment. In other embodiments, several residues are
sequentially added at one location before moving on to the next
location. Cycle times will generally be limited by the coupling
reaction rate, now as short as about 10 min in automated
oligonucleotide synthesizers. This step is optionally followed by
addition of a protecting group to stabilize the array for later
testing. For some types of polymers (e.g., peptides), a final
deprotection of the entire surface (removal of photoprotective side
chain groups) may be required.
More particularly, as shown in FIG. 2A, the glass 20 is provided
with regions 22, 24, 26, 28, 30, 32, 34, and 36. Regions 30, 32,
34, and 36 are masked, indicated by the hatched regions, as shown
in FIG. 2B and the glass is irradiated by the bright regions 22,
24, 26, and 28, and exposed to a reagent containing a
photosensitive blocked C (e.g., cytosine derivative), with the
resulting structure shown in FIG. 2C. The substrate is carefully
washed and the reactants removed. Thereafter, regions 22, 24, 26,
and 28 are masked, as indicated by the hatched region, the glass is
irradiated (as shown in FIG. 2D), as indicated by the bright
regions, at 30, 32, 34, and 36, and exposed to a photosensitive
blocked reagent containing T (e.g., thymine derivative), with the
resulting structure shown in FIG. 2E. The process proceeds,
consecutively masking and exposing the sections as shown until the
structure shown in FIG. 2M is obtained. The glass is irradiated and
the terminal groups are, optionally, capped by acetylation. As
shown, all possible trimers of cytosine/thymine are obtained.
In this example, no side chain protective group removal is
necessary, as might be common in modified nucleotides. If it is
desired, side chain deprotection may be accomplished by treatment
with ethanedithiol and trifluoroacetic acid.
In general, the number of steps needed to obtain a particular
polymer chain is defined by:
where:
n=the number of monomers in the basis set of monomers, and
l=the number of monomer units in a polymer chain.
Conversely, the synthesized number of sequences of length l will
be:
Of course, greater diversity is obtained by using masking
strategies which will also include the synthesis of polymers having
a length of less than 1. If, in the extreme case, all polymers
having a length less than or equal to 1 are synthesized, the number
of polymers synthesized will be:
The maximum number of lithographic steps needed will generally be n
for each "layer" of monomers, i.e., the total number of masks (and,
therefore, the number of lithographic steps) needed will be
n.times.l. The size of the transparent mask regions will vary in
accordance with the area of the substrate available for synthesis
and the number of sequences to be formed. In general, the size of
the synthesis areas will be:
size of synthesis areas=(A)/(S)
where:
A is the total area available for synthesis; and
S is the number of sequences desired in the area.
It will be appreciated by those of skill in the art that the above
method could readily be used to simultaneously produce thousands or
millions of oligomers on a substrate using the photolithographic
techniques disclosed herein. Consequently, the method results in
the ability to practically test large numbers of, for example, di,
tri, tetra, penta, hexa, hepta, octa, nona, deca, even
dodecanucleotides, or larger polynucleotides (or correspondingly,
polypeptides).
The above example has illustrated the method by way of a manual
example. It will of course be appreciated that automated or
semi-automated methods could be used. The substrate would be
mounted in a flow cell for automated addition and removal of
reagents, to minimize the volume of reagents needed, and to more
carefully control reaction conditions. Successive masks will be
applicable manually or automatically. See, e.g., Pirrung et al.
(1992) U.S. Pat. No. 5,143,854 and Ser. No. 07/624,120, now
abandoned.
7. Labeling of Target
The target oligonucleotide can be labeled using standard procedures
referred to above. As discussed, for certain situations, a reagent
which recognizes interaction, e.g., ethidium bromide, may be
provided in the detection step. Alternatively, fluorescence
labeling techniques may be applied, see, e.g., Smith, et al. (1986)
Nature, 321:674-679; and Prober, et al. (1987) Science,
238:336-341. The techniques described therein will be followed with
minimal modifications as appropriate for the label selected.
8. Dimers of A, C, G, and T
The described technique may be applied, with photosensitive blocked
nucleotides corresponding to adenine, cytosine, guanine, and
thymine, to make combinations of polynucleotides consisting of each
of the four different nucleotides. All 16 possible dimers would be
made using a minor modification of the described method.
9. 10-mers of A, C, G, and T
The described technique for making dimers of A, C, G, and T may be
further extended to make longer oligonucleotides. The automated
system described, e.g., in Pirrung et al. (1992) U.S. Pat. No.
5,143,854, and Ser. No. 07/624,120, now abandoned, can be adapted
to make all possible 10-mers composed of the 4 nucleotides A, C, G,
and T. The photosensitive, blocked nucleotide analogues have been
described above, and would be readily adaptable to longer
oligonucleotides.
10. Specific Recognition Hybridization to 10-mers
The described hybridization conditions are directly applicable to
the sequence specific recognition reagents attached to the
substrate, produced as described immediately above. The 10-mers
have an inherent property of hybridizing to a complementary
sequence. For optimum discrimination between full matching and some
mismatch, the conditions of hybridization should be carefully
selected, as described above. Careful control of the conditions,
and titration of parameters should be performed to determine the
optimum collective conditions.
11. Hybridization
Hybridization conditions are described in detail, e.g., in Hames
and Higgins (1985) Nucleic Acid Hybridisation: A Practical
Approach; and the considerations for selecting particular
conditions are described, e.g., in Wetmur and Davidson, (1988) J.
Mol. Biol. 31:349-370, and Wood et al. (1985) Proc. Natl. Acad.
Sci. USA 82:1585-1588. As described above, conditions are desired
which can distinguish matching along the entire length of the probe
from where there is one or more mismatched bases. The length of
incubation and conditions will be similar, in many respects, to the
hybridization conditions used in Southern blot transfers.
Typically, the GC bias may be minimized by the introduction of
appropriate concentrations of the alkylammonium buffers, as
described above.
Titration of the temperature and other parameters is desired to
determine the optimum conditions for specificity and
distinguishability of absolutely matched hybridization from
mismatched hybridization.
A fluorescently labeled target or set of targets are generated, as
described in Prober, et al. (1987) Science 238:336-341, or Smith,
et al. (1986) Nature 321:674-679. Preferably, the target or targets
are of the same length as, or slightly longer, than the
oligonucleotide probes attached to the substrate and they will have
known sequences. Thus, only a few of the probes hybridize perfectly
with the target, and which particular ones did would be known.
The substrate and probes are incubated under appropriate conditions
for a sufficient period of time to allow hybridization to
completion. The time is measured to determine when the probe-target
hybridizations have reached completion. A salt buffer which
minimizes GC bias is preferred, incorporating, e.g., buffer, such
as tetramethyl ammonium or tetraethyl ammonium ion at between about
2.4 and 3.0M. See Wood, et al. (1985) Proc. Nat'l Acad. Sci. USA
82:1585-1588. This time is typically at least about 30 min, and may
be as long as about 1-5 days. Typically very long matches will
hybridize more quickly, very short matches will hybridize less
quickly, depending upon relative target and probe concentrations.
The hybridization will be performed under conditions where the
reagents are stable for that time duration.
Upon maximal hybridization, the conditions for washing are
titrated. Three parameters initially titrated are time,
temperature, and cation concentration of the wash step. The matrix
is scanned at various times to determine the conditions at which
the distinguishability between true perfect hybrid and mismatched
hybrid is optimized. These conditions will be preferred in the
sequencing embodiments.
12. Positional Detection of Specific Interaction
As indicated above, the detection of specific interactions may be
performed by detecting the positions where the labeled target
sequences are attached. Where the label is a fluorescent label, the
apparatus described, e.g., in Pirrung et al. (1992) U.S. Pat. No.
5,143,854; and Ser. No. 07/624,120, now abandoned, may be
advantageously applied. In particular, the synthetic processes
described above will result in a matrix pattern of specific
sequences attached to the substrate, and a known pattern of
interactions can be converted to corresponding sequences.
In an alternative embodiment, a separate reagent which
differentially interacts with the probe and interacted
probe/targets can indicate where interaction occurs or does not
occur. A single-strand specific reagent will indicate where no
interaction has taken place, while a double-strand specific reagent
will indicate where interaction has taken place. An intercalating
dye, e.g., ethidium bromide, may be used to indicate the positions
of specific interaction.
13. Analysis
Conversion of the positional data into sequence specificity will
provide the set of subsequences whose analysis by overlap segments,
may be performed, as described above.
Analysis is provided by the methodology described above, or using,
e.g., software available from the Genetic Engineering Center, P.O.
Box 794, 11000 Belgrade, Yugoslavia (Yugoslav group). See, also,
Macevicz, PCT publication no. WO 90/04652, which is hereby
incorporated herein by reference.
B. Polypeptide
The description of the preparation of short peptides on a substrate
incorporates by reference sections in Pirrung et al. (1992) U.S.
Pat. No. 5,143,854, and described below.
1. Slide Preparation
Preparation of the substrate follows that described above for
nucleotides.
2. Linker Attachment, Blocking of Free Sites
The aminated surface of the slide is exposed to about 500 .mu.l of,
e.g., a 30 millimolar (mM) solution of NVOC-GABA (gamma amino
butyric acid) NHS (N-hydroxysuccinimide) in DMF for attachment of a
NVOC-GABA to each of the amino groups. The surface is washed with,
for example, DMF, methylene chloride, and ethanol. See Ser. No.
07,624,120, now abandoned, for details on amino acid chemistry.
Any unreacted aminopropyl silane on the surface, i.e., those amino
groups which have not had the NVOC-GABA attached, are now capped
with acetyl groups (to prevent further reaction) by exposure to a
1:3 mixture of acetic anhydride in pyridine for 1 hour. Other
materials which may perform this residual capping function include
trifluoroacetic anhydride, formicacetic anhydride, or other
reactive acylating agents. Finally, the slides are washed again
with DMF, methylene chloride, and ethanol.
3. Synthesis of 8 trimers of "A" and "B"
See Pirrung et al. (1992) U.S. Pat. No. 5,143,854 which describes
the preparation of glycine and phenylalanine trimers. The technique
is similar to the method described above for making triners of C
and T, but substituting photosensitive blocked glycine for the C
derivative and photosensitive blocked phenylalamine for the T
derivative.
4. Synthesis of a Dimer of an Aminopropyl Group and a Fluorescent
Group
In synthesizing the dimer of an aminopropyl group and a fluorescent
group, a functionalized Durapore.TM. membrane was used as a
substrate. The Durapore.TM. membrane was a polyvinylidine
difluoride with aminopropyl groups. The aminopropyl groups were
protected with the DDZ group by reaction of the carbonyl chloride
with the amino groups, a reaction readily known to those of skill
in the art. The surface bearing these groups was placed in a
solution of THF and contacted with a mask bearing a checkerboard
pattern of 1 mm opaque and transparent regions. The mask was
exposed to ultraviolet light having a wavelength down to at least
about 280 nm for about 5 minutes at ambient temperature, although a
wide range of exposure times and temperatures may be appropriate in
various embodiments of the invention. For example, in one
embodiment, an exposure time of between about 1 and 5000 seconds
may be used at process temperatures of between -70.degree. and
+50.degree. C.
In one preferred embodiment, exposure times of between about 1 and
500 seconds at about ambient pressure are used. In some preferred
embodiments, pressure above ambient is used to prevent
evaporation.
The surface of the membrane was then washed for about 1 hour with a
fluorescent label which included an active ester bound to a chelate
of a lanthanide. Wash times will vary over a wide range of values
from about a few minutes to a few hours. These materials fluoresce
in the red and the green visible region. After the reaction with
the active ester in the fluorophore was complete, the locations in
which the fluorophore was bound could be visualized by exposing
them to ultraviolet light and observing the red and the green
fluorescence. It was observed that the derivatized regions of the
substrate closely corresponded to the original pattern of the
mask.
5. Demonstration of Signal Capability
Signal detection capability was demonstrated using a low-level
standard fluorescent bead kit manufactured by Flow Cytometry
Standards and having model no. 824. This kit includes 5.8 .mu.m
diameter beads, each impregnated with a known number of fluorescein
molecules.
One of the beads was placed in the illumination field on the scan
stage in a field of a laser spot which was initially shuttered.
After being positioned in the illumination field, the photon
detection equipment was turned on. The laser beam was unblocked and
it interacted with the particle bead, which then fluoresced.
Fluorescence curves of beads impregnated with 7,000 and 29,000
fluorescein molecules, are shown in FIGS. 11A and 11B, respectively
of Pirrung et al. (1992) U.S. Pat. No. 5,143,854. On each curve,
traces for beads without fluorescein molecules are also shown.
These experiments were performed with 488 nm excitation, with 100
.mu.W of laser power. The light was focused through a 40 power 0.75
NA objective.
The fluorescence intensity in all cases started off at a high value
and then decreased exponentially. The fall-off in intensity is due
to photobleaching of the fluorescein molecules. The traces of beads
without fluorescein molecules are used for background subtraction.
The difference in the initial exponential decay between labeled and
nonlabeled beads is integrated to give the total number of photon
counts, and this number is related to the number of molecules per
bead. Therefore, it is possible to deduce the number of photons per
fluorescein molecule that can be detected. This calculation
indicates the radiation of about 40 to 50 photons per fluorescein
molecule are detected.
6. Determination of the Number of Molecules Per Unit Area
Aminopropylated glass microscope slides prepared according to the
methods discussed above were utilized in order to establish the
density of labeling of the slides. The free amino termini of the
slides were reacted with FITC (fluorescein isothiocyanate) which
forms a covalent linkage with the amino group. The slide is then
scanned to count the number of fluorescent photons generated in a
region which, using the estimated 40-50 photons per fluorescent
molecule, enables the calculation of the number of molecules which
are on the surface per unit area.
A slide with aminopropyl silane on its surface was immersed in a 1
mM solution of FITC in DMF for 1 hour at about ambient temperature.
After reaction, the slide was washed twice with DMF and then washed
with ethanol, water, and then ethanol again. It was then dried and
stored in the dark until it was ready to be examined.
Through the use of curves similar to those shown in FIG. 11 of
Pirrung et al. (1992) U.S. Pat. No. 5,143,854, and by integrating
the fluorescent counts under the exponentially decaying signal, the
number of free amino groups on the surface after derivitization was
determined. It was determined that slides with labeling densities
of 1 fluorescein per 10.sup.3 .times.10.sup.3 to .sup..about.
2.times.2 nm could be reproducibly made as the concentration of
aminopropyltriethoxysilane varied from 10.sup.-5 % to 10.sup.-1
%.
7. Removal of NVOC and Attachment of a Fluorescent Marker
NVOC-GABA groups were attached as described above. The entire
surface of one slide was exposed to light so as to expose a free
amino group at the end of the gamma amino butyric acid. This slide,
and a duplicate which was not exposed, were then exposed to
fluorescein isothiocyanate (FITC).
FIG. 12A of Pirrung et al. (1992) U.S. Pat. No. 5,143,854
illustrates the slide which was not exposed to light, but which was
exposed to FITC. The units of the x axis are time and the units of
the y axis are counts. The trace contains a certain amount of
background fluorescence. The duplicate slide was exposed to 350 nm
broadband illumination for about 1 minute (12 mW/cm.sup.2,
.sup..about. 350 nm illumination), washed and reacted with FITC. A
large increase in the level of fluorescence is observed, which
indicates photolysis has exposed a number of amino groups on the
surface of the slides for attachment of a fluorescent marker.
8. Use of a Mask in Removal of NVOC
The next experiment was performed with a 0.1% aminopropylated
slide. Light from a Hg--Xe arc lamp was imaged onto the substrate
through a laser-ablated chrome-on-glass mask in direct contact with
the substrate.
This slide was illuminated for approximately 5 minutes, with 12 mW
of 350 nm broadband light and then reacted with the 1 mM FITC
solution. It was put on the laser detection scanning stage and a
graph was plotted as a two-dimensional representation of position
color-coded for fluorescence intensity. The experiment was repeated
a number of times through various masks. The fluorescence patterns
for a 100.times.100 .mu.m mask, a 50 .mu.m mask, a 20 .mu.m mask,
and a 10 .mu.m mask indicate that the mask pattern is distinct down
to at least about 10 .mu.m squares using this lithographic
technique.
9. Attachment of YGGFL and Subsequent Exposure to Herz Antibody and
Goat Anti-Mouse Antibody
In order to establish that receptors to a particular polypeptide
sequence would bind to a surface-bound peptide and be detected, Leu
enkephalin was coupled to the surface and recognized by an
antibody. A slide was derivatized with 0.1% amino
propyl-triethoxysilane and protected with NVOC. A 500 .mu.m
checkerboard mask was used to expose the slide in a flow cell using
backside contact printing. The Leu enkephalin sequence (H.sub.2
N-tyrosine,glycine,glycine,phenylalanine,leucine-COOH, otherwise
referred to herein as YGGFL) was attached via its carboxy end to
the exposed amino groups on the surface of the slide. The peptide
was added in DMF solution with the BOP/HOBT/DIEA coupling reagents
and recirculated through the flow cell for 2 hours at room
temperature.
A first antibody, known as the Herz antibody, was applied to the
surface of the slide for 45 minutes at 2 .mu.g/ml in a
supercocktail (containing 1% BSA and 1% ovalbumin also in this
case). A second antibody, goat anti-mouse fluorescein conjugate,
was then added at 2 .mu.g/ml in the supercocktail buffer, and
allowed to incubate for 2 hours.
The results of this experiment were plotted as fluorescence
intensity as a function of position. This image was taken at 10
.mu.m steps and showed that not only can deprotection be carried
out in a well defined pattern, but also that (1) the method
provided for successful coupling of peptides to the surface of the
substrate, (2) the surface of a bound peptide was available for
binding with an antibody, and (3) the detection apparatus
capabilities were sufficient to detect binding of a receptor.
Moreover, the Herz antibody is a sequence specific reagent which
may be used advantageously as a sequence specific recognition
reagent. It may be used, if specificity is high, for sequencing
purposes, and, at least, for fingerprinting and mapping uses.
10. Monomer-By-Monomer Formation of YGGFL and Subsequent Exposure
to Labeled Antibody
Monomer-by-monomer synthesis of YGGFL and GGFL in alternate squares
was performed on a slide in a checkerboard pattern and the
resulting slide was exposed to the Herz antibody.
A slide is derivatized with the aminopropyl group, protected in
this case with t-BOC (t-butoxycarbonyl). The slide was treated with
TFA to remove the t-BOC protecting group. E-aminocaproic acid,
which was t-BOC protected at its amino group, was then coupled onto
the aminopropyl groups. The aminocaproic acid serves as a spacer
between the aminopropyl group and the peptide to be synthesized.
The amino end of the spacer was deprotected and coupled to
NVOC-leucine. The entire slide was then illuminated with 12 mW of
325 nm broadband illumination. The slide was then coupled with
NVOC-phenylalanine and washed. The entire slide was again
illuminated, then coupled to NVOC-glycine and washed. The slide was
again illuminated and coupled to NVOC-glycine to form the sequence
shown in the last portion of FIG. 13A of Pirrung et al. (1992) U.S.
Pat. No. 5,143,854.
Alternating regions of the slide were then illuminated using a
projection print using a 500.times.500 .mu.m checkerboard mask;
thus, the amino group of glycine was exposed only in the lighted
areas. When the next coupling chemistry step was carried out,
NVOC-tyrosine was added, and it coupled only at those spots which
had received illumination. The entire slide was then illuminated to
remove all the NVOC groups, leaving a checkerboard of YGGFL in the
lighted areas and in the other areas, GGFL. The Herz antibody
(which recognizes the YGGFL, but not GGFL) was then added, followed
by goat anti-mouse fluorescein conjugate.
The resulting fluorescence scan showed dark areas containing the
tetrapeptide GGFL, which is not recognized by the Herz antibody
(and thus there is no binding of the goat anti-mouse antibody with
fluorescein conjugate) , and red areas in which YGGFL was present.
The YGGFL pentapeptide is recognized by the Herz antibody and,
therefore, there is antibody in the lighted regions for the
fluorescein-conjugated goat anti-mouse to recognize.
Similar patterns for a 50 .mu.m mask used in direct contact
("proximity print") with the substrate provided a pattern which was
more distinct and the corners of the checkerboard pattern were
touching as a result of the mask being placed in direct contact
with the substrate (which reflects the increase in resolution using
this technique)
11. Monomer-By-Monomer Synthesis of YGGFL and PGGFL
A synthesis using a 50 .mu.m checkerboard mask was conducted.
However, P was added to the GGFL sites on the substrate, through an
additional coupling step. P was added by exposing protected GGFL to
light through a mask, and subsequence exposure to P in the manner
set forth above. Therefore, half of the regions on the substrate
contained YGGFL and the remaining half contained PGGFL.
The fluorescence plot for this experiment showed the regions are
again readily discernable between those in which binding did and
did not occur. This experiment demonstrated that antibodies are
able to recognize a specific sequence and that the recognition is
not length-dependent.
12. Monomer-By-Monomer Synthesis of YGGFL and YPGGFL
In order to further demonstrate the operability of the invention, a
50 .mu.m checkerboard pattern of alternating YGGFL and YPGGFL was
synthesized on a substrate using techniques like those set forth
above. The resulting fluorescence plot showed that the antibody was
clearly able to recognize the YGGFL sequence and did not bind
significantly at the YPGGFL regions.
13. Synthesis of an Array of Sixteen Different Amino Acid Sequences
and Estimation of Relative Binding Affinity to Herz Antibody
Using techniques similar to those set forth above, an array of 16
different amino acid sequences (replicated four times) was
synthesized on each of two glass substrates. The sequences were
synthesized by attaching the sequence NVOC-GFL across the entire
surface of the slides. Using a series of masks, two layers of amino
acids were then selectively applied to the substrate. Each region
had dimensions of 0.25 cm.times.0.0625 cm. The first slide
contained amino acid sequences containing only L- amino acids while
the second slide contained selected D- amino acids. Various regions
on the first and second slides, were duplicated four times on each
slide. The slides were then exposed to the Herz antibody and
fluorescein-labeled goat anti-mouse antibodies.
A fluorescence plot of the first slide, which contained only L-
amino acids showed red areas (indicating strong binding, i.e.,
149,000 counts or more) and black areas (indicating little or no
binding of the Herz antibody, i.e., 20,000 counts or less). The
sequence YGGFL was clearly most strongly recognized. The sequences
YAGFL and YSGFL also exhibited strong recognition of the antibody.
By contrast, most of the remaining sequences showed little or no
binding. The four duplicate portions of the slide were extremely
consistent in the amount of binding shown therein.
A fluorescence plot of the D- amino acid slide indicated that
strongest binding was exhibited by the YGGFL sequence. Significant
binding was also detected to YaGFL, YsGFL, and YpGFL. The remaining
sequences showed less binding with the antibody. Low binding
efficiency of the sequence yGGFL was observed.
Table 6 lists the various sequences tested in order of relative
fluorescence, which provides information regarding relative binding
affinity.
TABLE 6 ______________________________________ Apparent Binding to
Herz Ab L- a.a. Set D- a.a. Set
______________________________________ YGGFL YGGFL YAGFL YaGFL
YSGFL YsGFL LGGFL YpGFL FGGFL fGGFL YPGFL yGGFL LAGFL faGFL FAGFL
wGGFL WGGFL yaGFL fPGFL waGFL
______________________________________
14. Illustrative Alternative Embodiment
According to an alternative embodiment of the invention, the
methods provide for attaching to the surface a caged binding member
which, in its caged form, has a relatively low affinity for other
potentially binding species, such as receptors and specific binding
substances. Such techniques are more fully described in copending
application Ser. No. 404,920, filed Sep. 8, 1989, and incorporated
herein by reference for all purposes. See also Ser. No. 07/435,316,
now abandoned, and Barrett et al. (1993) U.S. Pat. No. 5,252,743,
each of which is hereby incorporated herein by reference.
According to this alternative embodiment, the invention provides
methods for forming predefined regions on a surface of a solid
support, wherein the predefined regions are capable of immobilizing
receptors. The methods make use of caged binding members attached
to the surface to enable selective activation of the predefined
regions. The caged binding members are liberated to act as binding
members ultimately capable of binding receptors upon selective
activation of the predefined regions. The activated binding members
are then used to immobilize specific molecules such as receptors on
the predefined region of the surface. The above procedure is
repeated at the same or different sites on the surface so as to
provide a surface prepared with a plurality of regions on the
surface containing, for example, the same or different receptors.
When receptors immobilized in this way have a differential affinity
for one or more ligands, screenings and assays for the ligands can
be conducted in the regions of the surface containing the
receptors.
The alternative embodiment may make use of novel caged binding
members attached to the substrate. Caged (unactivated) members have
a relatively low affinity for receptors of substances that
specifically bind to uncaged binding members when compared with the
corresponding affinities of activated binding members. Thus, the
binding members are protected from reaction until a suitable source
of energy is applied to the regions of the surface desired to be
activated. Upon application of a suitable energy source, the caging
groups labilize, thereby presenting the activated binding member. A
typical energy source will be light.
Once the binding members on the surface are activated they may be
attached to a receptor. The receptor chosen may be a monoclonal
antibody, a nucleic acid sequence, a drug receptor, etc. The
receptor will usually, though not always, be prepared so as to
permit attaching it, directly or indirectly, to a binding member.
For example, a specific binding substance having a strong binding
affinity for the binding member and a strong affinity for the
receptor or a conjugate of the receptor may be used to act as a
bridge between binding members and receptors if desired. The method
uses a receptor prepared such that the receptor retains its
activity toward a particular ligand.
Preferably, the caged binding member attached to the solid
substrate will be a photoactivatable biotin complex, i.e., a biotin
molecule that has been chemically modified with photoactivatable
protecting groups so that it has a significantly reduced binding
affinity for avidin or avidin analogs than does natural biotin. In
a preferred embodiment, the protecting groups localized in a
predefined region of the surface will be removed upon application
of a suitable source of radiation to give binding members, that is
biotin or a functionally analogous compound having substantially
the same binding affinity for avidin or avidin analogs as does
biotin.
In another preferred embodiment, avidin or an avidin analog is
incubated with activated binding members on the surface until the
avidin binds strongly to the binding members. The avidin so
immobilized on predefined regions of the surface can then be
incubated with a desired receptor or conjugate of a desired
receptor. The receptor will preferably be biotinylated, e.g., a
biotinylated antibody, when avidin is immobilized on the predefined
regions of the surface. Alternatively, a preferred embodiment will
present an avidin/biotinylated receptor complex, which has been
previously prepared, to activated binding members on the
surface.
II. FINGERPRINTING
The above section on generation of reagents for sequencing provides
specific reagents useful for fingerprinting applications.
Fingerprinting embodiments may be applied towards polynucleotide
fingerprinting, polypeptide fingerprinting, cell and tissue
classification, cell and tissue temporal development stage
classification, diagnostic tests, forensic uses for individual
identification, classification of organisms, and genetic screening
of individuals. Mapping applications are also described below.
A. Polynucleotide Fingerprint
Polynucleotide fingerprinting may use reagents similar to those
described above for probing a sequence for the presence of specific
subsequences found therein. Typically, the subsequences used for
fingerprinting will be longer than the sequences used in
oligonucleotide sequencing. In particular, specific long segments
may be used to determine the similarity of different samples of
nucleic acids. They may also be used to fingerprint whether
specific combinations of information are provided therein.
Particular probe sequences are selected and attached in a
positional manner to a substrate. The means for attachment may be
either using a caged biotin method described, e.g., in Barrett et
al. (1993) U.S. Pat. No. 5,242,743, or by another method using
targeting molecules. For example, a short polypeptide of specific
sequence may be attached to an oligonucleotide and targeted to
specific positions on a substrate having antibodies attached
thereto, the antibodies exhibiting specificity for binding to those
short peptide sequences. In another embodiment, an unnatural
nucleotide or similar complementary binding molecule may be
attached to the fingerprinting probe and the probe thereby directed
towards complementary sequences on a VLSIPS substrate. Typically,
unnatural nucleotides would be preferred, e.g., unnatural optical
isomers, which would not interfere with natural nucleotide
interactions.
Having produced a substrate with particular fingerprint probes
attached thereto at positionally defined regions, the substrate may
be used in a manner quite similar to the sequencing embodiment to
provide information as to whether the fingerprint probes are
detecting the corresponding sequence in a target sequence. This
will often provide information similar to a Southern blot
hybridization.
3. Polypeptide Fingerprint
A polypeptide fingerprint may be performed using antibodies which
recognize specific antigens on the polypeptide. For example,
monoclonal antibodies which recognize specific sequences or
antigens on a polypeptide may be used to determine whether those
epitopes are found on a particular protein. For example, particular
patterns of epitopes would be found on various types of proteins.
This will lead to the discovery that specific epitopes, or
antigenic determinants, which are characteristic of, e.g., beta
sheet segments, will be identified as will particular different
types of domains in various protein types. Thus, a screening method
may be devised which can classify polypeptides, either native or
denatured, into various new classes defined by the epitopes
existing thereon.
In addition, once the substrate is generated in the manners
described above, a target peptide is exposed to the substrate. The
target may be either native or denatured, though the conditions
used to denature the polypeptide may interfere with the specific
interaction between the polypeptide and the recognition reagent.
This method is not dependent on the fact that the polypeptide is a
single chain, thus protein complexes may also be fingerprinted
using this methodology. Structures such as multi-subunit proteins,
associations of proteins, ribosomes, nucleosomes, and other small
cellular structures may also be fingerprinted and classified
according to the presence of specific recognizable features
thereon.
Peptide fingerprinting may be useful, for example, in correlating
with particular physiological conditions or developmental stages of
a cell or organism. Thus, a biological sample may be fingerprinted
to determine the presence in that sample of a plurality of
different polypeptides which are each individually fingerprinted.
In an alternative embodiment, a polypeptide itself is not
fingerprinted but a biological sample is fingerprinted searching
for specific epitopes, e.g., polypeptide, carbohydrate, nucleic
acid, or any of a number of other specific recognizable structural
features.
The conditions for the interactions using antibodies is described,
e.g., in Harlow and Lane (1988) Antibodies: A Laboratory Manual,
Cold Spring Harbor Press, New York. The conditions should be
titrated for temperature, buffer composition, tire, and other
important parameters in an antibody interaction.
C. Cell Classification Scheme
The present invention can be used for cell classification using
fingerprinting type technology as described above in the
polypeptide fingerprint. Classes of cells are typically defined by
the presence of common functions which are usually reflected by
structural features. Thus, a plant cell is classified differently
from an animal cell by a number of structural features. Given an
unknown cell, the present invention provides improved means for
distinguishing the different cell types. Once a cell classification
scheme is developed and the structural features which define it are
identified using the present invention, homogeneous cell population
expressing these features may be separated from others. Standard
cell sorters may be coupled with recognition reagents and labels
which can distinguish various cell types.
a. T-Cell Classes
T-cell classes are defined on the basis of expression of particular
antigens characteristic of each class. For example, mouse T-cell
differentiation markers include the LY antigens. With the plurality
of different antigens which may be tested using antibody or other
recognition reagents, new populations and classes of cells may be
defined. For example, different neural cell types may be defined on
the basis of cell surface antigens. Different tissue types will be
defined on the basis of tissue specific antigens. Developmental
cell classes will be similarly defined. All of these screenings can
make use of the VLSIPS substrates with specific recognition
molecules attached thereto. The substrates are exposed to the cell
types directly, assaying for attachment of cells to specific
regions, or are exposed to products of a population of cells, e.g.,
a supernatant, or a cell lysate.
Once a cell classification scheme has been correlated with specific
structural markers therein, reagents which recognize those features
may be developed and used in a fluorescence activated cell sorter
as described, e.g., in Dangl, J. and Herzenberg (1982) J.
Immunological Methods 52:1-14; and Becton Dickinson, Fluorescence
Activated Cell Sorters Division, San Jose, Calif. This will provide
a homogeneous population of cells whose function has been defined
by structure.
b. B-Cell Classes
The present cell classification scheme may also be used to
determine specific B-cell classes. For example, B-cells specific
for producing IgM, IgG, IgD, IgE, and IgA may be defined by the
internal expression of specific mRNA sequences encoding each type
of immunoglobulin. The classification scheme may depend on either
extracellularly expressed markers which are correlated as being
diagnostic of specific stages in development, or intracellular mRNA
sequences which indicate particular functions.
D. Temporal Development Scheme
1. Developmental Antigens
The present fingerprinting invention also allows cell
classification by expression of developmental antigens. For
example, a lymphocyte stem cell expresses a particular combination
of antigens. As the lymphocyte develops through a program
developmental scheme, at various stages it expresses particular
antigens which are diagnostic of particular stages in development.
Again, the fingerprinting methodology allows for the definition of
specific structural features which are diagnostic of developmental
or functional features which will allow classification of cells
into temporal developmental classes. Cells, products of those
cells, or lysates of those cells will be assayed to determine the
developmental stage of the source cells. In this manner, once a
developmental stage is defined, specific synchronized populations
of cells will be selected out of another population. These
synchronized populations may be very important in determining the
biological mechanisms of development.
2. Developmental mRNA Expression
Besides expressed antigens, the present invention also allows for
fingerprinting of the mRNA population of a cell. In this fashion,
the mRNA population, which should be a good determinant of
developmental stage, will be correlated with other structural
features of the cell. In this manner, cells at specific
developmental stages will be characterized by the intracellular
environment, as well as the extracellular environment. The present
invention also allows the combination of definitions based, in
part, upon antigens and, in part, upon mRNA expression.
In one embodiment, the two may be combined in a single incubation
step. A particular incubation condition may be found which is
compatible with both hybridization recognition and
non-hybridization recognition molecules. Thus, e.g., an incubation
condition may be selected which allows both specificity of antibody
binding and specificity of nucleic acid hybridization. This allows
simultaneous performance of both types of interactions on a single
matrix. Again, where developmental mRNA patterns are correlated
with structural features, or with probes which are able to
hybridize to intracellular mRNA populations, a cell sorter may be
used to sort specifically those cells having desired mRNA
population patterns.
E. Diagnostic Tests
The present invention also provides the ability to perform
diagnostic tests. Diagnostic tests typically are based upon a
fingerprint type assay, which tests for the presence of specific
diagnostic structural features. Thus, the present invention
provides means for viral strain identification, bacterial strain
identification, and other diagnostic tests using positionally
defined specific reagents. The present invention also allows for
determining a spectrum of allergies, diagnosing a biological sample
for any or all of the above, and testing for many other
conditions.
1. Viral Identification
The present invention provides reagents and methodology for
identifying viral strains. The specific reagents may be either
antibodies or recognition proteins which bind to specific viral
epitopes preferably surface exposed, but may make use of internal
epitopes, e.g., in a denatured viral sample. In an alternative
embodiment, the viral genome may be probed for specific sequences
which are characteristic of particular viral strains. As above, a
combination of the two may be performed simultaneously in a single
interaction step, or in separate tests, e.g., for both genetic
characteristics and epitope characteristics.
2. Bacterial Identification
Similar techniques will be applicable to identifying a bacterial
source. This may be useful in diagnosing bacterial infections, or
In classifying sources of particular bacterial species. For
example, the bacterial assay may be useful in determining the
natural range of survivability of particular strains of bacteria
across regions of the country or in different ecological
niches.
3. Other Microbiological Identifications
The present invention provides means for diagnosis of other
microbiological and other species, e.g., protozoal species and
parasitic species in a biological sample, but also provides the
means for assaying a combination of different infections. For
example, a biological specimen may be assayed for the presence of
any or all of these microbiological species. In human diagnostic
uses, typical samples will be blood, sputum, stool, urine, or other
samples.
4. Allergy Tests
An immobilized set of antigens may be attached to a solid substrate
and, instead of the standard skin reaction tests, a blood sample
may be assayed on such a substrate to determine the presence of
antibodies, e.g., IgE or other type antibodies, which may be
diagnostic of an allergic or immunological susceptibility. A
standard radioallergosorbent test (RAST) may be used to check a
much larger population of antigens.
In addition, an allergy like test may be used to diagnose the
immunological history of a particular individual. For example, by
testing the circulating antibodies in a blood sample, which
reflects the immunological history and memory of an individual, it
may be determined what infections may not have been historically
presented to the immune system. In this manner, it may be possible
to specifically supplement an immune system for a short period of
time with IgG fractions made up of sepecific types of gamma
globulins. Thus, hepatitis gamma globulin injections may be better
designed for a particular environment to which a person is expected
to be exposed. This also provides the ability to identify
genetically equivalent individuals who have immunologically
different experiences. Thus, a blood sample from an individual who
has a particular combination of circulating antibodies will likely
be different from the combination of circulating antibodies found
in a genetically similar or identical individual. This could allow
for the distinction between clones of particular animals, e.g.,
mice, rats, or other animals.
F. Individual Identification
The present invention provides the ability to fingerprint and
identify a genetic individual. This individual may be a bacterial
or lower microorganism, as described above in diagnostic tests, or
of a plant or animal. An individual may be identified genetically
or immunologically, as described.
1. Genetic
Genetic fingerprinting has been utilized in comparing different
related species in Southern hybridization blots. Genetic
fingerprinting has also been used in forensic studies, see, e.g.,
Morris et al. (1989) J. Forensic Science 34:1311-1317, and
references cited therein. As described above, an individual may be
identified genetically by a sufficiently large number of probes.
The likelihood that another individual would have an identical
pattern over a sufficiently large number of probes may be
statistically negligible. However, it is often quite important that
a large number of probes be used where the statistical probability
of matching is desired to be particularly low. In fact, the probes
will optimally be selected for having high heterogeneity among the
population. In addition, the fingerprint method may make use of the
pattern of homologies indicated by a series of more and more
stringent washes. Then, each position has both a sequence
specificity and a homology measurement, the combination of which
greatly increases the number of dimensions and the statistical
likelihood of a perfect pattern match with another genetic
individual.
2. Immunological
As indicated above in the diagnostic tests, it is possible to
identify a particular immune system within a genetically
homogeneous class of organisms by virtue of their immunological
history. For example, a large colony of cloned mice may be
distinguishable by virtue of each immunological history. For
example, one mouse may have had an immunological response to
exposure to antigen A to which her genetically identical sibling
may have not been exposed. By virtue of this differential history,
the first of the pair will likely have a high antibody titer
against the antigen A whereas her genetically identical sibling
will have not had a response to that antigen by virtue of never
having been exposed to it. For this reason, immune systems may be
identified by their immunological memories. Thus, immunological
experience may also be a means for identifying a particular
individual at a particular moment in her lifetime.
This same immunological screening may be used for other sorts of
identifiable biological products. For example, an individual may be
identified by her combination of expressed proteins. These proteins
may reflect a physiological state of the individual, and would thus
be useful in certain circumstances where diagnostic tests may be
performed. For example, an individual may be identified, in part,
by the presence of particular metabolic products.
In fact, a plant origin may be determined by virtue of having
within its genome an unnatural sequence introduced to it by genetic
breeders. Thus, a marker nucleic acid sequence may be introduced as
a means to determine whether a genetic strain of a plant or animal
originated from another particular source.
G. Genetic Screening
1. Test Alleles With Markers
The present invention provides for the ability to screen for
genetic variations of individuals. For example, a number of genetic
diseases are linked with specific alleles. See, e.g., Scriber, C.
et al. (eds.) (1989) The Metabolic Bases of Inherited Disease,
McGraw-Hill, New York. In one embodiment, cystic fibrosis has been
correlated with a specific gene, see, Gregory et al. (1990) Nature
347:382-386. A number of alleles are correlated with specific
genetic deficiencies. See, e.g., McKusick, V. (1990) Genetic
Inheritance in Man: Catalogs of Autosomal Dominant, Autosomal
Recessive, and X-linked Phenotypes, Johns Hopkins University Press,
Baltimore; Ott, J. (1985) Analysis of Human Genetic Linkage, Johns
Hopkins University Press, Baltimore; Track, R. et al. (1989)
Banbury Report 32: DNA Technology and Forensic Science, Cold Spring
Harbor Press, New York; each of which is hereby incorporated herein
by reference.
2. Amniocentesis
Typically, amniocentesis is used to determine whether chromosome
translocations have occurred. The mapping procedure may provide the
means for determining whether these translocations have occurred,
and for detecting particular alleles of various markers.
III. MAPPING
A. Positionally Located Clones
The present invention allows for the positional location of
specific clones useful for mapping. For example, caged biotin may
be used for specifically positioning a probe to a location on a
matrix pattern.
In addition, the specific probes may be positionally directed to
specific locations on a substrate by targeting. For example,
polypeptide specific recognition reagents may be attached to
oligonucleotide sequences which can be complementarily targeted to
specific locations on a VLSIPS.TM. Technology substrate.
Hybridization conditions, as applied for oligonucleotide probes,
will be used to target the reagents to locations on a substrate
having complementary oligonucleotides synthesized thereon. In
another embodiment, oligonucleotide probes may be attached to
specific polypeptide targeting reagents such as an antigen or
antibody. These reagents can be directed towards a complementary
antigen or antibody already attached to a VLSIPS substrate.
In another embodiment, an unnatural nucleotide which does not
interfere with natural nucleotide complementary hybridization may
be used to target oligonucleotides to particular positions on a
substrate. Unnatural optical isomers of natural nucleotides should
be ideal candidates.
In this way, short probes may be used to determine the mapping of
long targets or long targets may be used to map the position of
shorter probes. See, e.g., Craig et al. 1990 Nuc. Acids Res.
18:2653-2660.
B. Positionally Defined Clones
Positionally defined clones may be transferred to a new substrate
by either physical transfer or by synthetic means. Synthetic means
may involve either a production of the probe on the substrate using
the VLSIPS.TM. Technology synthetic methods, or may involve the
attachment of a targeting sequence made by VLSIPS synthetic methods
which will target that positionally defined clone to a position on
a new substrate. Both methods will provide a substrate having a
number of positionally defined probes useful in mapping.
IX. CONCLUSION
The present inventions provide greatly improved methods and
apparatus for synthesis of polymers on substrates. It is to be
understood that the above description is intended to be
illustrative and not restrictive. Many embodiments will be apparent
to those of skill in the art upon reviewing the above description.
By way of example, the invention has been described primarily with
reference to the use of photoremovable protective groups, but it
will be readily recognized by those of skill in the art that
sources of radiation other than light could also be used. For
example, in some embodiments it may be desirable to use protective
groups which are sensitive to electron beam irradiation, x-ray
irradiation, in combination with electron beam lithograph, or x-ray
lithography techniques. Alternatively, the group could be removed
by exposure to an electric current. The scope of the invention
should, therefore, be determined not with reference to the above
description, but should instead be determined with reference to the
appended claims, along with the full scope of equivalents to which
such claims are entitled.
All publications and patent applications referred to herein are
incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually
incorporated by reference. The present invention now being fully
described, it will be apparent to one of ordinary skill in the art
that many changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.
* * * * *